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Sample records for cell line mc3t3-e1

  1. Effects of irradiation on TGF-{beta}{sub 1} mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line

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    Song, Ju Seop; Kim, Kyoung A; Koh, Kwang Joon [Department of Oral and Maxillofacial Radiology, School of Dentistry, and Institute of Oral Bio Science, Chonbuk National University, Jeonju (Korea, Republic of)

    2008-09-15

    To investigate the effects of irradiation on transforming growth factor {beta}1 (TGF-{beta}{sub 1}) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Cells were cultured in alpha-minimum essential medium ({alpha}-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with {alpha}-MEM supplemented with 10% FBS, 5 mM {beta}-glycerol phosphate, and 50 {mu}g/mL ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-{beta}{sub 1} mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. The amount of TGF-{beta}{sub 1} mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy, and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P<0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-{beta}{sub 1} mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line

  2. Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells

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    Wu, Junyi

    Chitosan is a natural linear polysaccharide that has been well known for its applications in drug delivery system due to its unique physicochemical and biological properties. However, challenges still remain for it to become a fully realized therapeutic agent. In this study, we investigated the uptake of chitosan nanoparticles (CNP) under the ultrasound stimulation, using a model cell culture system (MC3T3-E1 mouse pre-osteoblasts). The CNP were fabricated by an ionic gelation method and were lyophilized prior to characterization and delivery to cells. Particle size and zeta potential were measured using Dynamic Light Scattering (DLS); the efficiency of chitosan complexation was measured using the ninhydrin assay. Cytotoxicity was examined by neutral red assay within 48 hours after delivery. The effect of ultrasound (US) on the efficiency of nanoparticle delivery to the MC3T3-E1 cells was examined at 1MHz and at either 1 or 2 W/cm2. Fluorescein isothiocyanate (FITC)-conjugated-CNP were used to visualize the internalized particles within the cytosol. The uptake of FITC-CNP exhibits a dose and time dependent effect, a strong FITC fluorescence was detected at the concentration of 500microg/mL under fluorescence microscope. Ultrasound assisted uptake of FITC-CNP performed a significant positive effect at 2W/cm2 with 60s of ultrasound exposure time. CNP displayed a slightly decrease in cell viability from 25microg/mL to 100microg/mL, while higher concentration of CNP facilitates the proliferation of MC3T3-E1 cells. Less than 10% of reduction in cell viability was observed for US at 1W/cm2 and 2W/cm2 with 30s and 60s of exposure time, which suggest a mild effect of US to MC3T3-E1 cell line.

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

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

    2015-01-01

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

  4. Green tea polyphenol (-)-epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3-E1 cells.

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    Kamon, Masayoshi; Zhao, Ran; Sakamoto, Kazuichi

    2009-12-16

    Recently, various physiological effects of the tea polyphenol catechin for alleviating diseases such as cancer, arteriosclerosis, hyperlipidaemia and osteoporosis have been reported. However, the physiological effect of catechin on bone metabolism remains unclear. We examined the physiological effect of EGCG [(-)-epigallocatechin-3-gallate], which is the main component of green tea catechin, on osteoblast development using the precursor cell line of osteoblasts, MC3T3-E1, and co-culture of the osteoblasts from mouse newborn calvaria and mouse bone marrow cells. Although EGCG did not affect the viability and proliferation of MC3T3-E1 cells, EGCG inhibited the osteoblast differentiation. Furthermore, EGCG did not affect the mineralization of differentiated MC3T3-E1 cells, and reduced osteoclast formation in co-culture. These results suggest that EGCG can effectively suppress bone resorption, and can be used as an effective medicine in the treatment of the symptoms of osteoporosis.

  5. Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.

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    Suh, K S; Choi, E M; Rhee, S Y; Kim, Y S

    2014-02-01

    Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca(2+) chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca(2+) release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.

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

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

    2014-08-01

    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.

  7. Comparison of the effects of elevated inorganic phosphate on primary human vascular smooth muscle cells and the pre-osteoblastic cell line MC3T3-E1

    DEFF Research Database (Denmark)

    Pedersen, Lasse Ebdrup

    the role of PiT1 in mesenchymal stem cell osteoblastic differentiation/mineralization and found that PiT1 is upstream of Runx2 expression in the osteoblastic differentiation also. While the role of PiT1 as a regulator of Pi-induced Runx2 expression in VSMCs has been interpreted as Pi causes an osteo......-/chondro-genic phenotypic shift in VSMCs, my research, however, suggest that not to be the case. Thus, the exact role of VSMCs in vascular mineralization still requires more research....

  8. Effects of 6-Hydroxyflavone on Osteoblast Differentiation in MC3T3-E1 Cells

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    Chien-Hung Lai

    2014-01-01

    Full Text Available Osteoblast differentiation plays an essential role in bone integrity. Isoflavones and some flavonoids are reported to have osteogenic activity and potentially possess the ability to treat osteoporosis. However, limited information concerning the osteogenic characteristics of hydroxyflavones is available. This study investigates the effects of various hydroxyflavones on osteoblast differentiation in MC3T3-E1 cells. The results showed that 6-hydroxyflavone (6-OH-F and 7-hydroxyflavone (7-OH-F stimulated ALP activity. However, baicalein and luteolin inhibited ALP activity and flavone showed no effect. Up to 50 μM of each compound was used for cytotoxic effects study; flavone, 6-OH-F, and 7-OH-F had no cytotoxicity on MC3T3-E1 cells. Moreover, 6-OH-F activated AKT and serine/threonine kinases (also known as protein kinase B or PKB, extracellular signal-regulated kinases (ERK 1/2, and the c-Jun N-terminal kinase (JNK signaling pathways. On the other hand, 7-OH-F promoted osteoblast differentiation mainly by activating ERK 1/ 2 signaling pathways. Finally, after 5 weeks of 6-OH-F induction, MC3T3-E1 cells showed a significant increase in the calcein staining intensity relative to merely visible mineralization observed in cells cultured in the osteogenic medium only. These results suggested that 6-OH-F could activate AKT, ERK 1/2, and JNK signaling pathways to effectively promote osteoblastic differentiation.

  9. Hierarchical polymeric scaffolds support the growth of MC3T3-E1 cells.

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    Akbarzadeh, Rosa; Minton, Joshua A; Janney, Cara S; Smith, Tyler A; James, Paul F; Yousefi, Azizeh-Mitra

    2015-02-01

    Tissue engineering makes use of the principles of biology and engineering to sustain 3D cell growth and promote tissue repair and/or regeneration. In this study, macro/microporous scaffold architectures have been developed using a hybrid solid freeform fabrication/thermally induced phase separation (TIPS) technique. Poly(lactic-co-glycolic acid) (PLGA) dissolved in 1,4-dioxane was used to generate a microporous matrix by the TIPS method. The 3D-bioplotting technique was used to fabricate 3D macroporous constructs made of polyethylene glycol (PEG). Embedding the PEG constructs inside the PLGA solution prior to the TIPS process and subsequent extraction of PEG following solvent removal (1,4-dioaxane) resulted in a macro/microporous structure. These hierarchical scaffolds with a bimodal pore size distribution (300 μm) contained orthogonally interconnected macro-channels generated by the extracted PEG. The diameter of the macro-channels was varied by tuning the dispensing parameters of the 3D bioplotter. The in vitro cell culture using murine MC3T3-E1 cell line for 21 days demonstrated that these scaffolds could provide a favorable environment to support cell adhesion and growth.

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

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

    2006-07-01

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

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

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

    2010-05-28

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

  12. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells.

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    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-04-08

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

  13. MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

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    Tohru Hayakawa

    2012-01-01

    Full Text Available The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm and sandblasting (Ra: approximately 1.0 μm, and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells.

  14. Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation.

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    Yu Wang

    Full Text Available Electrical stimulation (ES is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP, and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN. ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases.

  15. A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yue-juan QIN; Zhen-lin ZHANG; Lu-yang YU; Jin-wei HE; Ya-nan HOU; Tian-jin LIU; Jia-cai WU; Song-hua WU; Li-he GUO

    2006-01-01

    Aim: To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis. Methods: OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibro-blast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively. Results: Weak A20 expression was found in MC3T3-El cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P<0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P<0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis. Conclusion: We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.

  16. The pharmacological effects of morroniside and loganin isolated from Liuweidihuang Wan, on MC3T3-E1 cells.

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    Li, Manyu; Wang, Wei; Wang, Ping; Yang, Kun; Sun, Hui; Wang, Xijun

    2010-10-21

    Liuweidihuang wan (LW), initially a well-known formula for curing "wu chi wu ruan", is commonly used nowadays for clinical treatment of postmenopausal osteoporosis (PO), but the identity of the effective substance(s) remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the proliferation of MC3T3-E1 cells, but both susbtances could improve the activity of alkaline phosphatase (ALP), and increase the contents of collagen type I and osteocalcin. Simultaneously, the mRNA expression of caspase-3, capase-9, RANKL was down-regulated and that of bcl-2 was up-regulated, which partially explains the anti-osteoporosis mechanism in MC3T3-E1 cells. In conclusion, morroniside and loganin may directly promote the differentiation and inhibit the apoptosis of MC3T3-E1 cells, and accordingly indirectly reduce bone resorption, which makes them promising natural drugs leads for treating PO in the near future.

  17. The Pharmacological Effects of Morroniside and Loganin Isolated from Liuweidihuang Wan, on MC3T3-E1 Cells

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    Xijun Wang

    2010-10-01

    Full Text Available Liuweidihuang Wan (LW, initially a well-known formula for curing “wu chi wu ruan”, is commonly used nowadays for clinical treatment of Postmenopausal Osteoporosis (PO, but the identity of the effective substance(s remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the proliferation of MC3T3-E1 cells, but both susbtances could improve the activity of alkaline phosphatase (ALP, and increase the contents of collagen type I and osteocalcin. Simultaneously, the mRNA expression of caspase-3, capase-9, RANKL was down-regulated and that of bcl-2 was up-regulated, which partially explains the anti-osteoporosis mechanism in MC3T3-E1 cells. In conclusion, morroniside and loganin may directly promote the differentiation and inhibit the apoptosis of MC3T3-E1 cells, and accordingly indirectly reduce bone resorption, which makes them promising natural drugs leads for treating PO in the near future.

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

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

    2013-11-01

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

  19. Pre-osteoblastic MC3T3-E1 cells promote breast cancer growth in bone in a murine xenograft model

    Institute of Scientific and Technical Information of China (English)

    Thomas M. Bodenstine; Benjamin H. Beck; Xuemei Cao; Leah M. Cook; Aimen Ismai; J. Kent Powers; Andrea M. Mastro; Danny R. Welch

    2011-01-01

    The bones are the most common sites of breast cancer metastasis. Upon arrival within the bone microenvironment, breast cancer cells coordinate the activities of stromal cells, resulting in an increase in osteoclast activity and bone matrix degradation. In late stages of bone metastasis, breast cancer cells induce apoptosis in osteoblasts, which further exacerbates bone loss. However, in early stages, breast cancer cells induce osteoblasts to secrete inflammatory cytokines purported to drive tumor progression. To more thoroughly evaluate the role of osteoblasts in early stages of breast cancer metastasis to the bones, we used green fluorescent protein-labeled human breast cancer cell lines MDA-MB-231 and MDA-MB-435, which both induce osteolysis after intra-femoral injection in athymic mice, and the murine pre-osteoblastic cell line MC3T3-E1 to modulate osteoblast populations at the sites of breast cancer metastasis. Breast cancer cells were injected directly into the femur with or without equal numbers of MC3T3-E1 cells. Tumors grew significantly larger when co-injected with breast cancer cells and MC3T3-E1 cells than injected with breast cancer cells alone. Osteolysis was induced in both groups, indicating that MC3T3-E1 cells did not block the ability of breast cancer cells to cause bone destruction. MC3T3-E1 cells promoted tumor growth out of the bone into the extraosseous stroma. These data suggest that breast cancer cells and osteoblasts communicate during early stages of bone metastasis and promote tumor growth.

  20. Inhibitory effect of apocynin on methylglyoxal-mediated glycation in osteoblastic MC3T3-E1 cells.

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    Suh, Kwang Sik; Rhee, Sang Youl; Kim, Young Seol; Choi, Eun Mi

    2015-04-01

    Methylglyoxal (MG), a highly reactive metabolite of hyperglycemia, can enhance protein glycation, oxidative stress or inflammation. The present study investigated the effects of apocynin on the mechanisms associated with MG toxicity in osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with apocynin prevented the MG-induced protein glycation and formation of intracellular reactive oxygen species and mitochondrial superoxide in MC3T3-E1 cells. In addition, apocynin increased glutathione levels and restored the activity of glyoxalase I inhibited by MG. These findings suggest that apocynin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing the MG detoxification system. Apocynin treatment decreased the levels of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6 induced by MG. Additionally, the nitric oxide level reduced by MG was significantly increased by apocynin. These findings indicate that apocynin might exert its therapeutic effects via upregulation of glyoxalase system and antioxidant activity. Taken together, apocynin may prove to be an effective treatment for diabetic osteopathy.

  1. The Pharmacological Effects of Morroniside and Loganin Isolated from Liuweidihuang Wan, on MC3T3-E1 Cells

    OpenAIRE

    Xijun Wang; Hui Sun; Ping Wang; Kun Yang; Wei Wang; Manyu Li

    2010-01-01

    Liuweidihuang Wan (LW), initially a well-known formula for curing “wu chi wu ruan”, is commonly used nowadays for clinical treatment of Postmenopausal Osteoporosis (PO), but the identity of the effective substance(s) remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the pr...

  2. Neuropeptide Y1 Receptor Regulates Glucocorticoid-Induced Inhibition of Osteoblast Differentiation in Murine MC3T3-E1 Cells via ERK Signaling

    Directory of Open Access Journals (Sweden)

    Wei Yu

    2016-12-01

    Full Text Available High dose glucocorticoid (GC administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10−7 M dexamethasone (Dex, Y1 receptor shRNA interference, Y1 receptor agonist [Leu31, Pro34]-NPY, and antagonist BIBP3226. Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8 assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation.

  3. Neuropeptide Y1 Receptor Regulates Glucocorticoid-Induced Inhibition of Osteoblast Differentiation in Murine MC3T3-E1 Cells via ERK Signaling.

    Science.gov (United States)

    Yu, Wei; Zhu, Chao; Xu, Wenning; Jiang, Leisheng; Jiang, Shengdan

    2016-12-21

    High dose glucocorticoid (GC) administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor) is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10(-7) M dexamethasone (Dex), Y1 receptor shRNA interference, Y1 receptor agonist [Leu(31), Pro(34)]-NPY, and antagonist BIBP3226. Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK) as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK) abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation.

  4. Neuropeptide Y1 Receptor Regulates Glucocorticoid-Induced Inhibition of Osteoblast Differentiation in Murine MC3T3-E1 Cells via ERK Signaling

    Science.gov (United States)

    Yu, Wei; Zhu, Chao; Xu, Wenning; Jiang, Leisheng; Jiang, Shengdan

    2016-01-01

    High dose glucocorticoid (GC) administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor) is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10−7 M dexamethasone (Dex), Y1 receptor shRNA interference, Y1 receptor agonist [Leu31, Pro34]-NPY, and antagonist BIBP3226. Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK) as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK) abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation. PMID:28009825

  5. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    Institute of Scientific and Technical Information of China (English)

    Xing ZHONG; Ling-ling XIU; Guo-hong WEI; Yuan-yuan LIU; Lei SU; Xiao-pei CAO; Yan-bing LI; Hai-peng XIAO

    2011-01-01

    Aim: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.Methods: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.Results: Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezaflbrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARa inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 pmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast prolifera-tion.Conclusion: Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.

  6. Ethanolic extract of Actaea racemosa (black cohosh) potentiates bone nodule formation in MC3T3-E1 preosteoblast cells.

    Science.gov (United States)

    Chan, B Y; Lau, K S; Jiang, B; Kennelly, E J; Kronenberg, F; Kung, A W C

    2008-09-01

    Aceaea racemosa (formerly Cimicifuga racemosa, black cohosh, AR) extracts have been widely used as an alternative to hormonal replacement therapy for menopausal symptoms. Recent evidences suggest AR extracts are also effective in protecting against postmenopausal bone loss. To determine whether AR has any direct anabolic effect on osteoblasts, we investigated the ethanolic extract of AR on bone nodule formation in mouse MC3T3-E1 preosteoblast cells. AR did not stimulate osteoblast proliferation. Rather, at high doses of 1000 ng/mL for 48 h, AR suppressed (7.2+/-0.9% vs. control) osteoblast proliferation. At 500 ng/mL, a significant increase in bone nodule formation was seen with Von Kossa staining. Using quantitative PCR analysis, AR was shown to enhance the gene expression of runx2 and osteocalcin. Co-treatment with ICI 182,780, the selective estrogen receptor antagonist, abolished the stimulatory effect of AR on runx2 and osteocalcin gene induction, as well as on bone nodule formation in MC3T3-E1 cells. This is a first report of the direct effect of AR on enhancement of bone nodule formation in osteoblasts, and this action was mediated via an estrogen receptor-dependent mechanism. The results provide a scientific rationale at the molecular level for the claim that AR can offer effective prevention of postmenopausal bone loss.

  7. Effect of fibroblast growth factor 9 on Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; PEI Yu; XIA Wei-bo; XING Xiao-ping; MENG Xun-wu; ZHOU Xue-ying

    2007-01-01

    Background Fibroblast growth factor 9 (FGF9), expressed in brain, kidney and developing skeletal tissues, can physiologically inhibit endochondral ossification; but little is known about how FGF9 affects osteoblasts and its detailed regulatory mechanism. Here we examined the effect of FGF9 on the activity of the murine Runt-related transcription factor2 (Runx2) gene promoter in preosteoblast MC3T3-E1 and premyoblast C2C12 cells.Methods Plasmids containing the Runx2 promoter region were transfected into MC3T3-E1 and C2C12 cells and stably transfected cell lines were established. The method of luciferase reporter gene activation was used to examine the effects of FGF9 on the promoter activity.Results FGF9 (10 ng/ml) increased Runx2 promoter activity in MC3T3-E1 cells. When MC3T3-E1 cells were treated with FGF9 plus the various inhibitors or activator of the intracellular signaling transducation pathways, including 10μmol/L U0126 (the inhibitor of mitogen-activated protein kinase kinase), 10 μmol/L SB203580 (the inhibitor of p38/mitogen activated protein kinase), or 1 μmol/L C6 ceramide (an activator of mitogen activated protein kinase), the luciferase expression did not change significantly compared with that of the cells treated with FGF9 only. However, when C2C12 cells were treated with 10 ng/ml FGF9, Runx2 gene promoter activity first decreased and then increased over a period of 1 to 5 days. Among the above inhibitors, only U0126 (10 μmol/L) completely blocked the effects of FGF9 on Runx2 gene promoter activity.Conclusions Our data showed that FGF9 can affect Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells. The action of FGF9 appears to depend partly on the mitogen-activated protein kinase kinase/mitogen-activated protein kinase pathways in C2C12 cells.

  8. Surface modifications by gas plasma control osteogenic differentiation of MC3T3-E1 cells

    NARCIS (Netherlands)

    Barradas, A.M.C.; Lachmann, K.; Hlawacek, G.; Frielink, C.; Truckenmuller, R.K.; Boerman, O.C.; Gastel, van R.; Garritsen, H.S.P.; Thomas, M.; Moroni, L.; Blitterswijk, van C.A.; Boer, de J.

    2012-01-01

    Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical compositi

  9. Surface modifications by gas plasma control osteogenic differentiation of MC3T3-E1 cells.

    NARCIS (Netherlands)

    Barradas, A.M.; Lachmann, K.; Hlawacek, G.; Frielink, C.; Truckenmoller, R.; Boerman, O.C.; Gastel, R. van; Garritsen, H.; Thomas, M.; Moroni, L.; Blitterswijk, C. Van; Boer, J. den

    2012-01-01

    Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical compositi

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

    Directory of Open Access Journals (Sweden)

    G. Lucchetta

    2015-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Yamauchi Mika

    2007-11-01

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

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

    Science.gov (United States)

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

    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 (4h, 1d, 3d, 7d), and cell proliferation was assessed by MTT method at 1d, 3d, and 7d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1h. The polished sample was smooth (Sq: 1.8nm), and the blasted and HA coated samples were much rougher (Sq: 3.2μm and 7.8μm). Within 1d 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 3d and 7d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1d and 3d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7d. Protein adsorption on the HA coated samples was the highest at 1h. 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.

  13. Protective Effects of Cerium Oxide Nanoparticles on MC3T3-E1 Osteoblastic Cells Exposed to X-Ray Irradiation

    Directory of Open Access Journals (Sweden)

    Cuifen Wang

    2016-04-01

    Full Text Available Background/Aims: Exposure to ionizing radiation can result in bone damage, including decreased osteocyte number and suppressed osteoblastic activity. However, molecular mechanisms remain to be elucidated, and effective prevention strategies are still limited. This study was to investigate whether cerium oxide nanoparticles (CeO2 NP can protect MC3T3-E1 osteoblast-like cells from damaging effects of X-ray irradiation, and to study the underpinning mechanism(s. Methods: MC3T3-E1, a osteoblast-like cell line, was exposed to X-ray irradiation and treated with different concentration of CeO2 nanoparticles. The micronucleus frequency was counted under a fluorescence microscope. Cell viability was evaluated using MTT assay. The effects of irradiation and CeO2 nanoparticles on alkaline phosphatase activity and MC3T3-E1 mineralization were further assayed. Results: We found that the ratio of micronuclei to binuclei was dose-dependently increased with X-ray irradiation (from 2 to 6 Gy, but diminished with the increased concentration of CeO2 NP treatment (from 50 to 100 nM. Exposure to X-rays (6 Gy decreased cell viability, differentiation and the mineralization, but CeO2 NP treatment (100 nM attenuated the deteriorative effects of irradiation. Both intracellular reactive oxygen species (ROS production and extracellular H2O2 concentration were increased after X-ray irradiation, but reduced following CeO2 NP treatment. Similar to irradiation, exposure to H2O2 (10 µM elevated the frequency of micronuclei and diminished cell viability and mineralization, while these changes were ameliorated following CeO2 NP treatment. Conclusions: Taken together, our findings suggest that CeO2 nanoparticles exhibit astonishing protective effects on irradiation-induced osteoradionecrosis in MC3T3-E1 cells, and the protective effects appear to be mediated, at least partially, by reducing oxidative stress.

  14. Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells.

    Science.gov (United States)

    Vishnuprasad, Chethala N; Tsuchiya, Tomoko; Kanegasaki, Shiro; Kim, Joon Ho; Han, Sung Soo

    2014-05-01

    Osteoporosis is one of the major metabolic bone diseases and is among the most challenging noncommunicable diseases to treat. Although there is an increasing interest in identifying bioactive molecules for the prevention and management of osteoporosis, such studies principally focus only on differentiation and mineralization of osteoblasts or inhibition of osteoclast activity. Stimulation of osteoblast migration must be a promising osteoanabolic strategy for improved metabolic bone disease therapy. In this study, we show that an anthraquinone derivative, aurantio-obtusin, stimulated chemotactic migration of MC3T3-E1 osteoblast cells in a concentration-dependent manner. The use of a real-time chemotaxis analyzing system, TAXIScan, facilitated the evaluation of both velocity and directionality of osteoblast migration in response to the compound. Besides migration, the compound stimulated osteoblast differentiation and mineralization. Taken together, the data presented in this paper demonstrate that aurantio-obtusin is a promising osteoanabolic compound of natural origin with potential therapeutic applications in the prevention of osteoporosis and other metabolic bone diseases.

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

    Science.gov (United States)

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

    2014-10-01

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

  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. Effect of Thymosin beta4 on the Differentiation and Mineralization of MC3T3-E1 Cell on a Titanium Surface.

    Science.gov (United States)

    Jeong, Soon-Jeong; Jeong, Moon-Jin

    2016-02-01

    Osteoblasts are responsible for the synthesis of bone matrix through the secretion of collagenous and non-collagenous proteins with mineralization. Thymosin beta4 (Tbeta4) is an actin-sequestering peptide that is involved in the regulation of cell proliferation, differentiation and motility. A recent study reported that the inhibition of Tbeta4 mRNA synthesis strongly decreases the level of gene expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), osteonectin (ON) and collagen type I (Col I) with mineralization during differentiation in odontoblasts. Titanium (Ti) is used commonly as an implant material for dental implants, which have strong mechanical potential and good biocompatibility with bone. This study examined whether Tbeta4 can be a potential molecule for promoting the differentiation and mineralization of MC3T3-E1 cells on a Ti surface. Tbeta4 increased the viability of MC3T3-E1 cells during differentiation on Ti discs compared to that of the control. The expression of Tbeta4 mRNA and protein in the Tbeta4-treated MC3T3-E1 cells was higher than the control during differentiation on the Ti discs. In addition, Tbeta4 increased the formation of mineralization nodules and the mRNA expression of alkaline phosphatase (ALP), DSPP, dentin matrix protein1 (DMP1), BSP and Col I compared to that of the control in MC3T3-E1 cells during differentiation on Ti discs. From the results, Tbeta4 increased the viability and promoted the differentiation and mineralization of MC3T3-E1 cells on Ti discs. This highlights the potential use of Tbeta4 for increasing osseointegration through osteoblast differentiation and mineralization on Ti discs.

  18. In vitro mineralization of MC3T3-E1 osteoblast-like cells on collagen/nano-hydroxyapatite scaffolds coated carbon/carbon composites.

    Science.gov (United States)

    Cao, Sheng; Li, Hejun; Li, Kezhi; Lu, Jinhua; Zhang, Leilei

    2016-02-01

    Collagen/nano-hydroxyapatite (collagen/nHA) scaffolds were successfully prepared on carbon/carbon composites as bioactive films using the layer-by-layer coating method. Surface characterizations of collagen/nHA scaffolds were detected by scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Compressive strengths of the scaffolds were evaluated by a universal test machine. In vitro biological performances were determined using scaffolds seeded with MC3T3-E1 osteoblasts-like cells and cultured in mineralization medium for up to 21 days. In addition, cellular morphologies and several related gene expressions of MC3T3-E1 cells in the scaffolds were also evaluated. Chemical and morphological analysis showed that the scaffolds had uniform pore sizes and unified phase composition. Mechanical testing indicated that the collagen/nHA scaffolds had the highest compressive strength in 50% of strain condition when the proportion of collagen and nano-hydroxyapatite was 1:3. Cellular morphology observations and cytology tests indicated that MC3T3-E1 cells were adhered on these scaffolds and proliferated. SEM photographs and gene expressions showed that mineralized MC3T3-E1 cells and newly formed extra cellular matrix (ECM) filled up the pores of the scaffolds after the 3-week mineralization inducement. Nano-sized apatite particles were secreted from MC3T3-E1 cells and combined with the reconstructed ECM. Collectively, collagen/nHA scaffolds provided C/C composites with a biomimetic surface for cell adhesion, proliferation and mineralized extra cellular matrices formation.

  19. Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-b estradiol

    Institute of Scientific and Technical Information of China (English)

    Zhen-Zhen Shang; Xin Li; Hui-Qiang Sun; Guo-Ning Xiao; Cun-Wei Wang; Qi Gong

    2014-01-01

    Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (a-MEM) cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 1028 mol?L21 17-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity;thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes, of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-b)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large

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

    Science.gov (United States)

    Wang, Jiawei; de Boer, Jan; de Groot, Klaas

    2009-09-01

    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 proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. It was found that MC3T3-E1 cells cultured on the biomimetically deposited carbonate apatite coating demonstrated the greatest proliferation rate and the highest differentiation potential. Cells on the biomimetically deposited octacalcium phosphate coating had lower proliferation rate before day 7, but higher after that, than those on the electrolytically deposited carbonate apatite coating. There was no difference on the expression of early differentiation markers, that is, alkaline phosphatase activity and collagen content, between biomimetically deposited octacalcium phosphate and electrolytically deposited carbonate apatite coatings. However, higher expression of late differentiation markers, that is, osteocalcin and bone sialoprotein mRNA, was found on the biomimetically deposited octacalcium phosphate coating on day 14. These results suggest that the difference in in vitro osteoblast cell performance of calcium phosphate coatings might relate to their physicochemical properties. Biomimetic carbonate apatite coating is the most favorable surface for the proliferation and differentiation of MC3T3-E1 cells.

  1. Influence of Mechanical Force on Bone Matrix Proteins in Ovariectomised Mice and Osteoblast-like MC3T3-E1 Cells

    Science.gov (United States)

    ZHANG, MENG; ISHIKAWA, SHINTARO; INAGAWA, TOMOKO; IKEMOTO, HIDESHI; GUO, SHIYU; SUNAGAWA, MASATAKA; HISAMITSU, TADASHI

    2017-01-01

    Aim: To investigate the effect of mechanical stress on periostin and semaphorin-3A expression in a murine model of postmenopausal osteoporosis and in osteoblast-like MC3T3-E1 cells. Materials and Methods: Female mice were divided into three groups and treated with a sham operation, ovariectomy (OVX) or OVX plus treadmill training (OVX+Run). After 10 weeks, tibias were used for histological analysis. MC3T3-E1 cells were burdened by mechanical stress using a centrifuge or were treated with periostin, and the production of biologically-active semaphorin-3A was examined in vitro. Results: In OVX+Run group tibias, the number of tartrate-resistant acid phosphatase-positive osteoclasts was lower than in the OVX group, and the expression of periostin and semaphorin-3A was higher. In MC3T3-E1 cells, centrifugal stress significantly increased periostin and semaphorin-3A mRNA expression. Treatment with periostin increased the semaphorin-3A level. Conclusion: We speculate that mechanical load may increase periostin production in osteoblasts, and periostin may inhibit osteoclast differentiation by its effects on semaphorin-3A. Our results support the concept of a positive correlation between exercise and inhibition of osteoclasts in post-menopausal osteoporosis. PMID:28064225

  2. Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, T.; Igarashi, C.; Takeuchi, Y.; Harada, S.; Kikuchi, T.; Yamato, H.; Ogata, E. (Fourth Department of Internal Medicine, University of Tokyo School of Medicine (Japan))

    1991-01-01

    Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.

  3. Influence of heating and cyclic tension on the induction of heat shock proteins and bone-related proteins by MC3T3-E1 cells.

    Science.gov (United States)

    Chung, Eunna; Sampson, Alana Cherrell; Rylander, Marissa Nichole

    2014-01-01

    Stress conditioning (e.g., thermal, shear, and tensile stress) of bone cells has been shown to enhance healing. However, prior studies have not investigated whether combined stress could synergistically promote bone regeneration. This study explored the impact of combined thermal and tensile stress on the induction of heat shock proteins (HSPs) and bone-related proteins by a murine preosteoblast cell line (MC3T3-E1). Cells were exposed to thermal stress using a water bath (44°C for 4 or 8 minutes) with postheating incubation (37°C for 4 hours) followed by exposure to cyclic strain (equibiaxial 3%, 0.2 Hz, cycle of 10-second tensile stress followed by 10-second rest). Combined thermal stress and tensile stress induced mRNA expression of HSP27 (1.41 relative fold induction (RFI) compared to sham-treated control), HSP70 (5.55 RFI), and osteopontin (1.44 RFI) but suppressed matrix metalloproteinase-9 (0.6 RFI) compared to the control. Combined thermal and tensile stress increased vascular endothelial growth factor (VEGF) secretion into the culture supernatant (1.54-fold increase compared to the control). Therefore, combined thermal and mechanical stress preconditioning can enhance HSP induction and influence protein expression important for bone tissue healing.

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

    Science.gov (United States)

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

    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 24h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7days, 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.

  5. miRNA expression profile during fluid shear stress-induced osteogenic differentiation in MC3T3-E1 cells

    Institute of Scientific and Technical Information of China (English)

    MAI Zhi-hui; PENG Zhu-li; ZHANG Jing-lan; CHEN Lin; LIANG Huan-you; CAI Bin; AI Hong

    2013-01-01

    Background Mechanical stress plays an important role in the maintenance of bone homeostasis.Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone.This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress (FSS) is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells.Methods MC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm2 using a parallel plate flow system.After FSS treatment,cytoskeleton immunohistochemical staining and microRNAs (miRNAs) were detected immediately.Osteogenic gene expression and immunohistochemical staining for collagen type Ⅰ were tested at the 24th hour after treatment,alkaline phosphatase (ALP) activity assay was performed at 24th,48th,and 72th hours after FSS treatment,and Alizarin Red Staining was checked at day 12.Results One hour of FSS at 12 dyn/cm2 induced actin stress fiber formation and rearrangement,up-regulated osteogenic gene expression,increased ALP activity,promoted synthesis and secretion of type Ⅰ collagen,enhanced nodule formation,and promoted terminal differentiation in MC3T3-E1 cells.During osteogenic differentiation,expression levels of miR-20a,-21,-19b,-34a,-34c,-140,and-200b in FSS-induced cells were significantly down-regulated.Conclusion The short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be invovled in FSS-induced pre-osteoblast differentiation.

  6. 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

  7. Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

    Directory of Open Access Journals (Sweden)

    Gang Mei

    2014-04-01

    Full Text Available Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10−10 to 10−8 M. To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1 antagonists and Dickkopf-1 (DKK1 and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2, were measured using quantitative polymerase chain reaction (PCR. Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10−9 to 10−8 M significantly up-regulated the expressions of osteoblastic genes. SP (10−8 M also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1, as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10−8 M promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.

  8. The effects of brushite coating extract with strontium on the osteoblast-related factor expression of MC3T3-E1 cells%透钙磷石涂层浸提液掺锶对 MC3T3-E1细胞成骨相关因子表达的影响

    Institute of Scientific and Technical Information of China (English)

    臣娟; 梁永强; 韩东颖

    2016-01-01

    Objective:To study the effects of brushite coating with strontium on the osteoblast-related factor expression of MC3T3-E1 cells.Methods:Brushite coating on tinanium surface was prepared by electrochemical deposition and the extract of the coating with stronium at 0.1%,0.5% and 1% were prepared respectively.MC3T3-E1 cells were cultured with the extracts,cell prolifera-tion,ALP activity,bFGF and VEGF mRNA were examined by MTT assay,ALP staining and RT-PCR respectively.Results:Stronti-um containing extracts promoted the proliferation,increased the ALP activity and mRNA expression of bFGF and VEGF(P <0.05)of MC3T3 cells.Conclusion:Brushite extract containing strontium may promote osteogenic function of MC3T3-E1 cells.%目的:探讨透钙磷石与锶对成骨细胞的协同作用。方法:电化学沉积制备含透钙磷石涂层钛片,浸提法制备其浸提液,向配制好的浸提液中分别加入0.1%、0.5%和1%的锶。将 MC3T3-E1细胞置于掺锶的浸提液中进行培养,检测不同含锶量的透钙磷石涂层浸提液中 MC3T3-E1细胞活性、ALP 活性,RT-PCR 检测 bFGF 和 VEGF mRNA 表达。结果:含透钙磷石涂层钛片浸提中锶的浓度为0.1%、0.5%和1%时,培养的 MC3T3-E1细胞增殖率、ALP 活性、bFGF 和 VEGF mRNA 表达均高于无锶对照组(P <0.05)。结论:掺锶透钙磷石对 MC3T3-E1细胞成骨功能有促进作用。

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-01

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

  10. Carbonated apatites obtained by the hydrolysis of monetite: influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells.

    Science.gov (United States)

    Pieters, Ilse Y; Van den Vreken, Natasja M F; Declercq, Heidi A; Cornelissen, Maria J; Verbeeck, Ronald M H

    2010-04-01

    The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO(4), DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075 mol l(-1). Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24h and 7 days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9 wt.% and levels out to an acceptable value (+ or - 50% compared to the control) for carbonate contents between 6.9 and 16.1 wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.

  11. Effect of borate glass composition on its conversion to hydroxyapatite and on the proliferation of MC3T3-E1 cells.

    Science.gov (United States)

    Brown, Roger F; Rahaman, Mohamed N; Dwilewicz, Agatha B; Huang, Wenhai; Day, Delbert E; Li, Yadong; Bal, B Sonny

    2009-02-01

    Glasses containing varying amounts of B(2)O(3) were prepared by partially or fully replacing the SiO(2) in silicate 45S5 bioactive glass with B(2)O(3). The effects of the B(2)O(3) content of the glass on its conversion to hydroxyapatite (HA) and on the proliferation of MC3T3-E1 cells were investigated in vitro. Conversion of the glasses to HA in dilute (20 mM) K(2)HPO(4) solution was monitored using weight loss and pH measurements. Proliferation of MC3T3-E1 cells was determined qualitatively by assay of cell density at the glass interface after incubation for 1 day and 3 days, and quantitatively by fluorescent measurements of total DNA in cultures incubated for 4 days. Higher B(2)O(3) content of the glass increased the conversion rate to HA, but also resulted in a greater inhibition of cell proliferation under static culture conditions. For a given mass of glass in the culture medium, the inhibition of cell proliferation was alleviated by using glasses with lower B(2)O(3) content, by incubating the cell cultures under dynamic rather than static conditions, or by partially converting the glass to HA prior to cell culture.

  12. The fast track to canonical Wnt signaling in MC3T3-E1 cells protected by substance P against serum deprivation-induced apoptosis.

    Science.gov (United States)

    Yang, Jianguo; Nie, Jiping; Fu, Su; Liu, Song; Wu, Jianqun; Cui, Liang; Zhang, Yongtao; Yu, Bin

    2017-01-01

    The canonical Wnt pathway is vital to bone physiology by increasing bone mass through elevated osteoblast survival. Although investigated extensively in stem cells, its role in osteoblastic MC3T3-E1 cells has not been completely determined. To explore how this pathway is regulated by different conditions, we assessed the anti-apoptotic effects of substance P on the canonical Wnt pathway in MC3T3-E1 cells by treating cells with serum deprivation or serum starving with "substance P," a neuropeptide demonstrated to promote bone growth and stimulate Wnt signaling. The results showed that serum deprivation both induced apoptosis and activated Wnt signal transduction while substance P further stimulated the Wnt pathway via the NK-1 receptor but protected the cells from apoptotic death. Fast-tracking of Wnt signaling by substance P was also noted. These results indicate that nutritional deprivation and substance P synergistically activated the canonical Wnt pathway, a finding that helps to reveal the role of Wnt signaling in bone physiology affected by nutritional deprivation and neuropeptide substance P.

  13. Influence of sulfonylureas on autophagy, apoptosis, and differentiation of the mice MC3T3-E1 cells%磺脲类药物对成骨细胞MC3T3-E1自噬、凋亡和分化功能的影响

    Institute of Scientific and Technical Information of China (English)

    张丽; 季虹; 苏华; 刘兴艳; 辛衍代; 荣海钦

    2013-01-01

    目的 探讨磺脲类药物对成骨细胞自噬、凋亡及分化功能的影响. 方法 用磺酰罗丹明B染色检测不同浓度的格列本脲(GLB)、格列齐特(GLC)和格列吡嗪(GLP)对成骨细胞存活率的影响;Western blot分析3种磺脲类药物对细胞中自噬、凋亡标志蛋白表达的变化;Hoechst染色镜下观察上述药物对细胞凋亡的影响;通过对骨钙素(OCN)和碱性磷酸酶(ALP)的测定研究药物对细胞分化功能的影响. 结果 中、高浓度的GLB、GLC和GLP使MC3T3-E1细胞存活率降低.在药物干预下,MC3T3-E1细胞自噬和凋亡标志蛋白表达增加,mTOR信号途径无明显变化.3种药物可使细胞分泌OCN和ALP的能力下降. 结论 GLB、GLC和GLP可诱导成骨细胞MC3T3-E1发生自噬和凋亡,降低成骨细胞分化功能,且可能通过mTOR-非依赖途径诱导细胞自噬.%Objective To investigate the influence of sulfonylureas on the autophagy, apoptosis, and differentiation of the MC3T3-E1 cells. Methods Sulforhodamine B Assay was carried out to determine the effect of glibenclamide, gliclazide and glipizide in different concentrations on the cell survival. Western blot analysis was done to determine the protein levels of autophagy and apoptosis markers. Hoechst staining was applied to observe the impact of the three sulfonylureas on the cell apoptosis. The osteocalcin and alkaline phosphatase were assayed to assess the influence of these drugs on the differentiation of MC3T3-E1 cells. Results The intervention with glibenclamide, gliclazide, and glipizide in high concentrations made the survival rate of MC3T3-E1 cells decreased. Under the condition of drug intervention, the expression of autophagy and apoptosis markers of MC3T3-E1 cells was enhanced, whereas no remarkable changes were detected in mTOR signaling. The three sulfonylureas weakened the secretion of osteocalcin and alkaline phosphatase of MC3T3-E1 cells. Conclusion Glbenclamide, gliclazide, and glipizide

  14. 饥饿素对贫铀所致MC3T3-E1细胞损伤的保护作用研究%Protective role of ghrelin in depleted uranium-induced damage of MC3 T3-E1 cells

    Institute of Scientific and Technical Information of China (English)

    郝玉徽; 黄嘉伟; 刘聪; 李蓉

    2015-01-01

    Objective To evaluate the impact of ghrelin on depleted uranium ( DU)-induced damage of the osteoblast MC3T3-E1. Methods MC3T3-E1 cells were treated with different doses of ghrelin for 1 h before DU (500 μM) treatment. After 24 hours,the cell via-bility,intracellular tartrate-resistant acid phosphatase (StrACP),alkaline phosphatase (AKP),osteoprotegerin (OPG),solvable receptor acti-vator of nuclear factor-κB ligand ( sRANKL) ,catalase ( CAT) and reactive oxygen species ( ROS) were measured. Results After DU expo-sure,ghrelin pretreatment increased the cell viability and CAT levels,and reduced intracellular StrACP,AKP,sRANKL/OPG and ROS in a dose-dependent manner. Conclusion Through maintaining the balance of OPG/RANKL and reducing the oxidative stress,ghrelin could pro-tect against DU-induced damage of MC3T3-E1 cells.%目的 评价饥饿素对贫铀(DU)所致成骨细胞(MC3T3-E1细胞)损伤的影响. 方法 不同浓度的饥饿素提前1 h预处理MC3T3-E1细胞后暴露于DU(500 μM)24 h,检测细胞存活率、细胞内抗酒石酸酸性磷酸酶(StrACP)、碱性磷酸酶(AKP)、骨保护素(OPG)、可溶性核因子-κB受体活化因子配体(sRANKL)含量以及细胞内过氧化氢酶(CAT)和活性氧(ROS)水平. 结果饥饿素预处理可明显提高DU暴露后细胞存活率及CAT含量,降低细胞内StrACP、AKP、sRANKL/OPG以及ROS水平,并且存在剂量依赖关系. 结论 饥饿素通过调节OPG/RANKL系统的失衡以及降低细胞内氧化应激,发挥对DU暴露后MC3T3-E1细胞的保护作用.

  15. 珍珠粉对 MC3T3-E1细胞增殖、分化、矿化及骨相关基因表达的影响%Effect of pearl powder on the proliferation, differentiation, and mineralization of MC3T3-E1 cells and the expression of bone related genes in the cells

    Institute of Scientific and Technical Information of China (English)

    王凯; 魏博; 谭佳妮; 季晖

    2014-01-01

    Objective To investigate the effect of pearl powder on the osteogenic proliferation, differentiation, interstitial mineralization, and apoptosis in MC3T3-E1 cells, and to explore the potential mechanisms.Methods MC3T3-E1 cells were cultured with pearl powder or low-calcium pearl powder in vitro.The osteogenic proliferation was determined using MTT method. And the differentiation was determined using alkaline phosphatase ( ALP) kit.The formation of mineralized nodules was observed using von Kossa staining. The mRNA expression of Runt-related transcription factor II ( Runx2 ) , osteocalcin ( OC ) , osteoprotegerin ( OPG ) , and receptor activator of NK-κB ligand ( RANKL ) was detected using RT-PCR.The cell apoptosis induced by serum deprivation was detected using flow cytometry.Results In a certain range of concentration, pearl powder and low-calcium pearl powder could significantly increase the secretion of ALP and the formation of mineralized nodules, and markedly improve the mRNA expression of Runx2 and OC in MC3T3-E1cells.Furthermore, pearl powder and low calcium pearl powder could significantly decrease the RANKL/OPG ratio, and ameliorate the apoptosis of the osteoblasts induced by serum deprivation. Conclusion Both pearl powder and low calcium pearl powder can promote the differentiation and maturation in MC3T3-E1cells. And the effect of pearl powder is much stronger, suggesting a critical role of calcium contained in pearl powder in regulating the osteogenic proliferation and differentiation in MC3T3-E1cells.%目的:研究珍珠粉对MC3T3-E1细胞成骨增殖、分化、间质矿化及凋亡的影响,并初步探讨其作用机制。方法在体外培养的MC3T3-E1细胞中分别加入珍珠粉和低钙珍珠粉,采用MTT法及碱性磷酸酶(Alkaline phosphatase,ALP)试剂盒检测其对MC3T3-E1细胞增殖及分化的影响;Von Kossa染色法观察矿化结节的形成;RT-PCR检测细胞中转录因子( Runt-related transcription factor

  16. 新型根管封闭剂对MC3T3-E1成骨细胞的细胞毒性评估%Cytotoxicity of New Root Canal Sealers on MC3T3 -E1 Osteoblast Cells.

    Institute of Scientific and Technical Information of China (English)

    孙燕; 樊明文; 李宇红; 陆瑶伽

    2011-01-01

    Objective: To investgate the cytotoxicity of new root canal sealers, RealSeal Sealer and GuttaFlow, on MC3T3 - El osteoblast cells and compare with traditional material AH Plus. Methods: Six samples of each sealer were fabricated in sterile cylindrical Teflon tubes and were kept in a humid chamber at 37 ℃ for 7 days. Then extraction of the specimens was obtained after incubating with cell culture medium for 3 day. MC3T3 -E1 cells were cultured with different concentrations of the extracts (100% , 50%, 25%, and 12. 5%) , and the cell relative proliferation rate was evaluate by CCK -8 assay at 24h and 72 h . Results: RealSeal Sealer showed more cytotoxicity to MC3T3 -E1 cells than AH Plus and GuttaFlow(P<0. 05) within 3 days. The cytotoxicity of GuttaFlow was similar to AH Plus and the negative control. Conclusion: RealSeal Sealer had a strong cytotoxicity on MC3T3-E1cells, while GuttaFlow and AH Plus showed almost nontoxicity.%目的:评估新型根管封闭剂RealSeal sealer、GuttaFlow对MC3T3- E1成骨细胞的细胞毒性,并与传统的AH Plus比较.方法:制备RealSeal sealer、GuttaFlow和AH Plus(固化7d)的材料浸提液,倍比稀释为4种浓度:100%、50%、25%、12.5%;MC3T3-E1细胞于其中分别培养24h和72h,CCK-8法检测细胞存活率,评价不同材料的细胞毒性.结果:在实验期内,RealSeal sealer组的细胞存活率显著低于AH Plus、GuttaFlow(P<0.05),AHPlus、GuttaFlow和阴性对照组间无显著差异.结论:在本实验条件下,RealSeal sealer对MC3T3一E1成骨细胞的毒性最强,而GuttaFlow和AH Plus几乎无细胞毒性.

  17. 辐射对体外培养MC3T3-E1成骨前体细胞增殖和分化的影响%Effects of irradiation on proliferation and differentiation of MC3T3-E1 osteoblastic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    李玉梅; 赵铱民; 查年保; 舒震; 张松

    2014-01-01

    Objective The aim of this study is to investigate the effects of irradiation on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Methods MC3T3-E1 cells were irradiated 24 h after initial seeding. Gamma-radiation was administered at 0, 4, and 8 Gy as single doses by using a 60Co source. Cell proliferation was assessed at days 1, 3, 5, and 7 post-irradiation by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide assay. The collagen secretion of the cells was measured through sirius red staining at day 12 post-irradiation. The expressions of osteogenesis-related genes were assessed through real time fluorescence quantitative polymerase chain reaction at day 16 post-irradiation. The matrix mineralization caused by cells was evaluated through alizarin red staining at day 28 post-irradiation. Results The cells exposed to 4 Gy or 8 Gy demonstrated significantly lower proliferation rates compared with the non-irradiated group. Doses of 4 Gy or more significantly inhibited the expressions of osteogenesis-related genes (Osterix and osteocalcin). Collagen secretion and cell mineralization were significantly reduced by the 8 Gy dose. Conclusion 60Co γ-rays dose-dependently suppress the proliferation, collagen secretion, and mineralization of MC3T3-E1 cells. Furthermore, radiation seems to dose-dependently inhibit the expressions of osteogenesis-related genes of the cells.%目的:观察60Coγ射线对MC3T3-E1成骨前体细胞增殖和分化能力的影响。方法 MC3T3-E1细胞接种24 h后进行60Coγ射线照射,单次照射剂量分别为0、4、8 Gy。辐射后第1、3、5、7天,采用噻唑蓝比色法检测细胞的增殖能力;辐射后第12天,用黏胶纤维红染色法检测细胞的胶原分泌情况;第16天,用实时荧光定量聚合酶链反应检测细胞成骨相关基因mRNA的表达;第28天,采用茜素红染色及定量分析检测细胞基质的矿化能力。结果与对照组(0 Gy)相比,4

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

    Directory of Open Access Journals (Sweden)

    Masaaki Takechi

    2016-04-01

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

  19. Fractal dimension as a measure of altered actin cytoskeleton in MC3T3-E1 cells under simulated microgravity using 3-D/2-D clinostats.

    Science.gov (United States)

    Qian, A R; Li, D; Han, J; Gao, X; Di, S M; Zhang, W; Hu, L F; Shang, Peng

    2012-05-01

    Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

  20. The effect of gold nanoparticles on the proliferation and differentiation of murine osteoblast: a study of MC3T3-E1 cells in vitro.

    Science.gov (United States)

    Yao, Yuanyuan; Shi, Xiujuan; Chen, Fengshan

    2014-07-01

    The current study involves in identification and molecular levels characterization of optimal size and concentration of gold nanoparticles (AuNPs). Stable, gold nanoparticles were synthesized and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The concentration and size dependent effects of the gold nanoparticles on proliferation of pre-osteoblast cells MC3T3-E1 was evaluated employing MTT cell proliferation assay. The results revealed that 30 nm diameter gold nanoparticles at a concentration of 10(-11) ppm were the most effective in promoting cell proliferation. Assay for alkaline phosphatase (ALP) activity and ALP staining were also used to confirm the effect of gold nanoparticles on osteoblast proliferation and differentiation. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the expression of the osteogenic genes Runx2, ALP, OCN and OPN as response gold nanoparticles. The data demonstrated that 30 nm gold nanoparticles at a concentration of 10(-11) ppm was the best combination of size and concentration to promote the proliferation and differentiation of osteoblasts, as indicated by an increase in the ALP activity and expression of the osteogenic genes Runx2, ALP, OCN and OPN. Collectively the results of this study suggest that gold nanoparticles can promote the proliferation and differentiation of osteoblasts and could be used effectively in treatments promoting bone regeneration.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-01

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

  2. 富血小板纤维蛋白提取液对高糖状态下MC3T3-E1细胞的影响%The effects of platelet-rich fibrin extract(PRFe) on MC3T3-E1 cells in the environment of different concentration glucose

    Institute of Scientific and Technical Information of China (English)

    陈涛; 王中; 刘振华; 高攀

    2015-01-01

    目的:探讨富血小板纤维蛋白提取液(Platelet-rich fibrin extract, PRFe)对不同高糖状态下MC3T3-E1细胞增殖、成骨分化的影响。方法:实验分为对照组(5.5mmol/L糖浓度培养基),高糖组(15.5mmol/L糖浓度培养基)及实验组(含PRFe的15.5mmol/L糖浓度培养基)。甲基噻唑基四唑(MTT)法测定1d,3d,5d的细胞增殖数;碱性磷酸酶(ALP)活性检测1d,3d,5d,7d的成骨分化情况;荧光实时定量PCR测定核心结合蛋白因子2(Runt-related transcription factor-2, RUNX2)和成骨细胞特异性转录因子(Osterix, OSX)基因分别在3d、7d的表达。结果:随培养时间延长,实验组细胞的增殖、分化及成骨基因的表达均高于高糖组,且差异具有统计学意义(P0.05)。结论: PRFe能有效地促进高糖状态下MC3T3-E1细胞的增殖、分化及成骨基因的表达。%Objective: To evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and bone differentiation of MC3T3-E1 cells in different concentration glucose environment. Methods: Trials are divided into the control group (5.5mmol/L), the high glucose group (15.5mmol/L)and the experimental group (15.5mmol/L+PRFe), the experimental group use the osteogenic induction α-MEM containing PRFe, while the other two groups just use the osteogenic induction α-MEM without PRFe. MTT assay to detect the number of the osteoblasts at 1d、3d、5d; the activity of alkaline phosphatase(ALP) to detect the differentiation of osteoblast at 1d、3d、5d、7d; mean while, the level of osteogenetic biomarkers Runx2 and OSX at 3d、7d were quantified by real-time PCR. Results: The number、ALP activity and the gene expression level of MC3T3-E1 cells was higher in the experiment group than high glucose group(P0.05). Conclu-sions: Our work confirmed that PRFe is useful in stimulating the proliferation and bone differentiation of MC3T3-E1 cells in the high glucose

  3. Proliferation and osteogenic response of MC3T3-E1 pre-osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Mygdali, Evdokia; Prymak, Oleg; Buyakov, Ales; Kulkov, Sergei; Chatzinikolaidou, Maria

    2015-11-01

    Dense zirconia ceramics are used in bone applications due to their mechanical strength and biocompatibility, but lack osseointegration. A porous interface in contact with bone tissue may lead to better bone bonding but the biological properties of porous zirconia are not widely explored. The present study focuses on the manufacturing of an yttria- (YSZ) and a magnesia-stabilized (MgSZ) porous zirconia, and on their in vitro biological investigation. The sintered ceramics had similar characteristics of porosity, pore size and interconnectivity. Their elastic moduli and compressive strength values were within the range of the values of human cortical bone. MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen deposition and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2 and Alp. Nevertheless, the biomineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts toward a mature state capable of mineralizing the extracellular matrix.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yumei; Zhao Yimin [School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Chai Feng; Hildebrand, Hartmut F [Groupe de Recherche sur les Biomateriaux, Faculte de Medecine, F-59045 Lille cedex (France); Hornez, Jean-Christophe [Laboratoire des Materiaux et Procedes (LMP), EA 2443, UVHC, 59600 Maubeuge (France); Li, Chang Liang [Northwest Institute for Nonferrous Metal Research, Xi' an 710016 (China); Traisnel, Michel, E-mail: zhaoym@fmmu.edu.c, E-mail: fhildebrand@univ-lille2.f [Ecole Nationale Superieure de Chimie de Lille, UMR CNRS 8008, 59652 Villeneuve d' Ascq (France)

    2009-02-15

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

  5. Effect of zinc on gene expression in osteoblastic MC3T3-E1 cells: enhancement of Runx2, OPG, and regucalcin mRNA expressions.

    Science.gov (United States)

    Yamaguchi, Masayoshi; Goto, Maya; Uchiyama, Satoshi; Nakagawa, Taeko

    2008-05-01

    The effect of zinc sulfate on the mRNA expressions in Runx2, osteocalcin, alpha1(I) collagen, insulin-like growth factor-I (IGF-I), transforming growth factor-beta1 (TGF-beta1), osteoprotegerin (OPG), regucalcin, zinc transporter 1 (ZIP1), or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) in osteoblastic MC3T3-E1 cells in vitro was investigated. Cells with subconfluency were cultured for 48 h in a medium containing either vehicle or zinc sulfate (10(-6)-10(-4) M) without fetal bovine serum. Culture with zinc sulfate (10(-5) M) caused a significant increase in Runx2, OPG, or regucalcin mRNA expressions in the cells, while it did not have a significant effect on osteocalcin, alpha1(I) collagen, IGF-I, TGF-beta1, ZIP1, or G3PDH mRNA expressions. The effect of zinc sulfate (10(-4) M) in increasing Runx2 mRNA expression was seen at 24-72 h after culture. A significant increase in OPG mRNA expression was observed at 24 or 48 h after culture. Regucalcin mRNA expression was significantly increased at 48 or 72 h after culture with zinc sulfate (10(-4) M). The stimulatory effects of zinc sulfate on Runx2, OPG, or regucalcin mRNAs were significantly prevented in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (10(-6) M), an inhibitor of transcription activity. Culture with beta-alanyl-L-histidinato zinc (10(-5) M) caused a significant increase in Runx2 or regucalcin mRNA expressions, while zinc acexamate (10(-5) M) did not have a significant effect on Runx2, OPG, ZIP1, or regucalcin mRNA expressions. This study demonstrates that zinc sulfate has a role in the enhancement of Runx2, OPG, or regucalcin mRNA expression in osteoblastic cells in vitro, suggesting its role in the regulation of gene expression in the cells.

  6. Glabridin Alleviates the Toxic Effects of Methylglyoxal on Osteoblastic MC3T3-E1 Cells by Increasing Expression of the Glyoxalase System and Nrf2/HO-1 Signaling and Protecting Mitochondrial Function.

    Science.gov (United States)

    Choi, Eun Mi; Suh, Kwang Sik; Kim, Yu Jin; Hong, Soo Min; Park, So Yong; Chon, Suk

    2016-01-13

    Methylglyoxal (MG) contributes to the pathogenesis of age- and diabetes-associated complications. The present study investigated the effects of glabridin on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. MC3T3-E1 cells were treated with glabridin in the presence of MG, and markers of mitochondrial function and oxidative damage were examined. Pretreatment of MC3T3-E1 osteoblastic cells with glabridin prevented MG-induced cell death, the production of intracellular reactive oxygen species and mitochondrial superoxides, cardiolipin peroxidation, and the production of inflammatory cytokines. The soluble form of receptor for advanced glycation end products (sRAGEs)/RAGE ratio increased upon MG treatment, but less so after pretreatment with glabridin, which also increased the level of reduced glutathione and the activities of glyoxalase I and heme oxygenase-1, all of which were reduced by MG. In addition, glabridin elevated the level of nuclear factor erythroid 2-related factor 2. These findings suggest that glabridin protects against MG-induced cell damage by inhibiting oxidative stress and increasing MG detoxification. Pretreatment of MC3T3-E1 osteoblastic cells with glabridin reduced MG-induced mitochondrial dysfunction. Additionally, the nitric oxide level significantly increased upon glabridin pretreatment. Together, these data show that glabridin may potentially serve to prevent the development of diabetic bone disease associated with MG-induced oxidative stress.

  7. Combined Effects of Androgen and Growth Hormone on Osteoblast Marker Expression in Mouse C2C12 and MC3T3-E1 Cells Induced by Bone Morphogenetic Protein

    Science.gov (United States)

    Kimura, Kosuke; Terasaka, Tomohiro; Iwata, Nahoko; Katsuyama, Takayuki; Komatsubara, Motoshi; Nagao, Ryota; Inagaki, Kenichi; Otsuka, Fumio

    2017-01-01

    Osteoblasts undergo differentiation in response to various factors, including growth factors and steroids. Bone mass is diminished in androgen- and/or growth hormone (GH)-deficient patients. However the functional relationship between androgen and GH, and their combined effects on bone metabolism, remains unclear. Here we investigated the mutual effects of androgen and GH on osteoblastic marker expression using mouse myoblastic C2C12 and osteoblast-like MC3T3-E1 cells. Combined treatment with dihydrotestosterone (DHT) and GH enhanced BMP-2-induced expression of Runx2, ALP, and osteocalcin mRNA, compared with the individual treatments in C2C12 cells. Co-treatment with DHT and GH activated Smad1/5/8 phosphorylation, Id-1 transcription, and ALP activity induced by BMP-2 in C2C12 cells but not in MC3T3-E1 cells. The insulin-like growth factor (IGF-I) mRNA level was amplified by GH and BMP-2 treatment and was restored by co-treatment with DHT in C2C12 cells. The mRNA level of the IGF-I receptor was not significantly altered by GH or DHT, while it was increased by IGF-I. In addition, IGF-I treatment increased collagen-1 mRNA expression, whereas blockage of endogenous IGF-I activity using an anti-IGF-I antibody failed to suppress the effect of GH and DHT on BMP-2-induced Runx2 expression in C2C12 cells, suggesting that endogenous IGF-I was not substantially involved in the underlying GH actions. On the other hand, androgen receptor and GH receptor mRNA expression was suppressed by BMP-2 in both cell lines, implying the existence of a feedback action. Collectively the results showed that the combined effects of androgen and GH facilitated BMP-2-induced osteoblast differentiation at an early stage by upregulating BMP receptor signaling. PMID:28067796

  8. Effects of 2-deoxy-D-glucose and quercetin on the gene expression of bone sialoprotein and osteocalcin during the differentiation in irradiated MC3T3-E1 osteoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Un; Kim, Kyoung A; Koh, Kwang Jun [Department of Oral and Maxillofacial Radiology, School of Dentistry, and Institute of Oral Bioscience, Chonbuk National University, Jeonju (Korea, Republic of)

    2009-09-15

    To investigate the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of bone sialoprotein (BSP) and osteocalcin (OC) during the differentiation in irradiated MC3T3-E1 osteoblastic cells. When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or 10 {mu}M QCT, and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the synthesis of type I collagen, and expression of BSP and OC. The synthesis of type I collagen in cells exposed to 2 Gy of radiation in the presence of 2-DG or QCT showed no significant difference compared with the control group within 15 days post-irradiation. When the cells were irradiated with 8 Gy, 2-DG facilitated the irradiation mediated decrease of type I collagen synthesis, whereas such decrease was inhibited by treating with QCT. During MC3T3-E1 osteoblastic cell differentiation, the mRNA expression of BSP and OC showed the peak value at 14 days and 21 days, respectively. 2-DG or QCT treatment alone decreased the level of BSP mRNA, but increased the OC mRNA level only at early time of differentiation (day 7). In the cells irradiated with 2, 4, 8 Gy, the mRNA expression of BSP and OC decreased at 7 days after the irradiation. The cells were treated with various dose of radiation in the presence of 2-DG or QCT, the mRNA level of both BSP and OC increased although this increase was observed at low dose of radiation (2 Gy) and at the early stage of differentiation. However, when the cells were exposed to 4, 6, or 8 Gy, the increase of BSP and OC mRNAs was detected only in cells co-incubated with QCT. This study demonstrates that 2-DG and QCT affect differently the expression of bone formation related factors, type I collagen, BSP, and OC in the irradiated MC3T3-E1 osteoblasic cells, according to the dose of radiation and the times of differentiation. Overall, the present findings

  9. 鱿鱼皮胶原蛋白水解肽对镉抑制MC3T3-E1增殖、分化及钙化的影响%Effect of Collagen Peptide Extracted from Dosidicus gigas Skin on Proliferation, Differentiation and Calcification of MC3T3-E1 Cell Induced by Cd

    Institute of Scientific and Technical Information of China (English)

    蔡江佳; 李晔; 全晶晶; 蔺佳良; 张云云; 王峰; 苏秀榕

    2015-01-01

    目的:探究秘鲁鱿鱼皮胶原蛋白水解肽增强MC3T3-E1细胞抗骨质疏松的作用.方法:将培养的MC3T3-E1细胞分为正常对照组、氯化镉损伤组和胶原蛋白水解肽干预组;利用MTT法确定氯化镉的半数抑制浓度,建立细胞损伤模型;根据各组细胞增殖率差异,确定最佳胶原蛋白水解肽的添加量;通过细胞周期、凋亡,碱性磷酸酶活性的测定及Alizarin red染色法分析胶原蛋白水解肽在细胞增殖、分化、钙化阶段拮抗氯化镉的抑制作用.结果:与氯化镉损伤组相比,胶原蛋白水解肽干预组细胞活性升高(P<0.01);处于G1期的细胞数量减小(P<0.05),S期细胞数量增大(P<0.05);凋亡率降低(P<0.05);9,12,15 d时AKP活性升高(P<0.05,P<0.01,P<0.01).结论:摄入一定剂量的氯化镉会抑制MC3T3-E1成骨细胞增殖、分化和钙化.胶原蛋白水解肽在一定程度上可改善此类损伤对MC3T3-E1细胞的影响.

  10. Protective Effects of Collagen Extracted from Dosidicus gigas Skin on MC3T3-E1 Cell Induced by H2O2%鱿鱼皮胶原蛋白对H2O2诱导MC3T3-E1损伤的修复作用

    Institute of Scientific and Technical Information of China (English)

    蔡江佳; 李晔; 张云云; 童茜茜; 王峰; 苏秀榕

    2015-01-01

    目的:探究秘鲁鱿鱼皮胶原蛋白增强MC3T3-E1细胞抗自由基损伤的作用机制.方法:将培养的MC3T3-E1细胞分为正常对照组、H2O2损伤组和胶原蛋白干预组;利用MTT法确定H2O2的半数抑制浓度,建立自由基损伤的细胞模型,通过试剂盒测定SOD活性和MDA含量来确定最佳的胶原蛋白添加量;在倒置显微镜下观察各分组的细胞形态;用流氏细胞仪检测细胞凋亡率,并分析细胞增殖周期;实时荧光定量RT-PCR测定HSP70和Bax的表达水平.结果:与H2O2损伤组相比,胶原蛋白干预组SOD活性升高(P<0.05),MDA含量降低(P<0.05),凋亡率降低(P<0.05),G0-G1期细胞百分率减小(P<0.05),S期细胞百分率增大(P<0.05),Bax表达减弱,HSP70表达增强(P<0.05).结论:摄入一定剂量的H2O2会导致MC3T3-E1成骨细胞的氧化损伤并促进其凋亡,而胶原蛋白可以改善此类损伤对MC3T3-E1细胞的影响.

  11. 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.

  12. Wnt/β-catenin signaling plays an important role in the protective effects of FDP-Sr against oxidative stress induced apoptosis in MC3T3-E1 cell.

    Science.gov (United States)

    Qi, Huan-Huan; Bao, Jun; Zhang, Qi; Ma, Bo; Gu, Gui-Ying; Zhang, Peng-Ling; Ou-Yang, Gang; Wu, Zi-Mei; Ying, Han-Jie; Ou-Yang, Ping-Kai

    2016-10-01

    Strontium fructose 1,6-diphosphate (FDP-Sr) is a new strontium-containing compound. The primary aim of this study was to clarify whether the structure component of FDP-Sr, FDP could benefit the protective effect of Sr (II) against oxidative stress induced apoptosis, and meanwhile to further explore the important role of Wnt/β-catenin signaling in the anti-apoptosis effect of FDP-Sr in response to oxidative stress induced by H2O2 in an osteoblastic MC3T3-E1 cell line. Results showed that FDP-Sr could improve the osteoblastic differentiation under oxidative stress with induced cell proliferation and improved mineralization. The inhibition effect of FDP-Sr on cell apoptosis induced by H2O2 was proved by reduced reactive oxygen species production and activated caspase3. Under oxidative stress, mRNA and protein levels of phospho-β-catenin reduced, while β-catenin increased in the FDP-Sr treatment cell, leaded to the up-regulations of Runx2 and OPG at both mRNA and protein levels, finally improved the differentiation of osteoblasts. By the engagement of Wnt/β-catenin pathway's inhibitor (XAV-939), the protective effects of FDP-Sr on osteoblastic differentiation against oxidative stress were repressed along with inhibited wnt/β-catenin signaling and reduced mRNA and protein levels of Runx2 and OPG. In conclusion, FDP-Sr was demonstrated to protect osteoblast differentiation from oxidative damage induced by H2O2 through up-regulation of Wnt/β-catenin signaling, and FDP in FDP-Sr was able to directly improve the oxidative stress injury through its ROS scavenging ability.

  13. Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction.

    Directory of Open Access Journals (Sweden)

    Sepideh Mostofi

    Full Text Available This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd were analysed by scanning electron microscopy (SEM and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for

  14. Pre-osteoblastic MC3T3-E1 promote breast cancer cell growth in bone in a murine xenograft model

    Science.gov (United States)

    The bones are the most common sites of breast cancer metastasis. Upon arrival within the bone microenvironment, breast cancer cells coordinate the activities of stromal cells, resulting in an increase in osteoclast activity and bone matrix degradation. In late stages of bone metastasis, breast cance...

  15. Mechanical loading induced expression of bone morphogenetic protein-2,alkaline phosphatase activity,and collagen synthesis in osteoblastic MC3T3-E1 cells

    Institute of Scientific and Technical Information of China (English)

    LU Hong-fei; MAI Zhi-hui; XU Ye; WANG Wei; AI Hong

    2012-01-01

    Background Bone morphogenetic protein(BMP)-2,alkaline phosphatase(ALP),and collagen typeⅠ?are known to play a critical role in the process of bone remodeling.However,the relationship between mechanical strain and the expression of BMP-2,ALP,and COL-Ⅰ?in osteoblasts was still unknown.The purpose of this study was to investigate the effects of different magnitudes of mechanical strain on osteoblast morphology and on the expression of BMP-2,ALP,and COL-Ⅰ.Methods Osteoblast-like cells were flexed at four deformation rates(0,6%,12%,and 18% elongation).The expression of BMP-2 mRNA,ALP,and COL-Ⅰ?in osteoblast-like cells were determined by real-time quantitative reverse transcription polymerase chain reaction,respectively.The results were subjected to analysis of variance(ANOVA)using SPSS 13.0 statistical software.Results The cells changed to fusiform and grew in the direction of the applied strain after the mechanical strain was loaded.Expression level of the BMP-2,ALP,and COL-Ⅰ?increased magnitude-dependently with mechanical loading in the experimental groups,and the 12% elongation group had the highest expression(P<0.05).Conclusion Mechanical strain can induce morphological change and a magnitude-dependent increase in the expression of BMP-2,ALP,and COL-Ⅰ?mRNA in osteoblast-like cells,which might influence bone remodeling in orthodontic treatment.

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

    Science.gov (United States)

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

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

  17. 透钙磷石涂层浸提液掺锶对MC3T3-E1细胞VEGF、βFGF和Runx2的影响%Influences of extract of brushite coating mixed with strontium on cells VEGF, βFGC and Runx2 in MC3T3-E1

    Institute of Scientific and Technical Information of China (English)

    文臣娟; 梁永强; 赵坚; 韩东颖

    2015-01-01

    目的 通过体外细胞学实验对透钙磷石与锶二者协同作用于小鼠成骨细胞进行相关研究,为优化锶浓度提供理论依据.方法 利用电化学沉积法制备含透钙磷石涂层的钛片,再利用浸提方法制备不同含锶量的浸提液.将MC3T3-E1细胞于浸提液中进行培养,利用RT-PCR技术检测各组细胞因子:血管生长因子(VEGF)、成纤维细胞生长因子β(βFGF)和侏儒蛋白相关转录因子2(Runx2)的表达情况.结果 透钙磷石对成骨标志物的表达有促进作用,而掺锶透钙磷石则更能促进这一作用.其与锶的浓度有关,当锶浓度为0.5%时其对成骨细胞的作用最明显.结论 透钙磷石具有良好的生物活性,具有良好的诱导成骨细胞分裂、分化的作用.而向透钙磷石中掺入不同浓度的锶,可以在一定程度上促进细胞VEGF、βFGF和Runx2的表达,从而明显增强其对成骨细胞分化的诱导作用.

  18. MC3T3-E1 Cell Response to Ti1-xAgx and Ag-TiNx Electrodes Deposited on Piezoelectric Poly(vinylidene fluoride) Substrates for Sensor Applications.

    Science.gov (United States)

    Marques, S M; Rico, P; Carvalho, I; Gómez Ribelles, J L; Fialho, L; Lanceros-Méndez, S; Henriques, M; Carvalho, S

    2016-02-17

    In the sensors field, titanium based coatings are being used for the acquisition/application of electrical signals from/to piezoelectric materials. In this particular case, sensors are used to detect dynamic mechanical loads at early stages after intervention of problems associated with prostheses implantation. The aim of this work is to select an adequate electrode for sensor applications capable, in an initial stage to avoid bone cell adhesion, but at a long stage, permit osteointegration and osteoinduction. This work reports on the evaluation of osteoblast MC3T3-E1 cells behavior in terms of proliferation, adhesion and long-term differentiation of two different systems used as sensor electrodes: Ti1-xAgx and Ag-TiNx deposited by d.c. and pulsed magnetron sputtering at room temperature on poly(vinylidene fluoride) (PVDF). The results indicated an improved effect of Ag-TiNx electrodes compared with Ti1-xAgx and TiN, in terms of diminished cell adhesion and proliferation at an initial cell culture stage. Nevertheless, when cell culture time is longer, cells grown onto Ag-TiNx electrodes are capable to proliferate and also differentiate at proper rates, indicating the suitability of this coating for sensor application in prostheses devices. Thus, the Ag-TiNx system was considered the most promising electrode for tissue engineering applications in the design of sensors for prostheses to detect dynamic mechanical loads.

  19. Comparison of alkaline phosphatase activity of MC3T3-E1 cells cultured on different Ti surfaces: modified sandblasted with large grit and acid-etched (MSLA), laser-treated, and laser and acid-treated Ti surfaces

    Science.gov (United States)

    Li, Lin-Jie; Kim, So-Nam

    2016-01-01

    PURPOSE In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P.05). CONCLUSION This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. PMID:27350860

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

    Science.gov (United States)

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

    2016-10-01

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

  1. 脉冲电磁场对MC3 T3-E1细胞增殖与分化的影响%Effect of pulsed electromagnetic fields on proliferation and differentiation of MC3 T3-E1 cells

    Institute of Scientific and Technical Information of China (English)

    闫娟丽; 李少锋; 周建; 葛宝丰; 陈克明; 马小妮; 方清清; 成魁; 高玉海; 石文贵

    2015-01-01

    目的:研究不同强度50Hz脉冲电磁场(Pulse electromagnetic fields, PEMFs)对小鼠成骨细胞系MC3T3⁃E1增殖与分化的影响。方法 MC3T3⁃E1传代培养后分成七组,分别采用0�0mT、0�6mT、1�2mT、1�8mT、2�4mT、3�0mT和3�6mT50Hz脉冲电磁场处理,90 min/d。 MTT法检测细胞增殖情况,成骨性分化检测处理3 d、6 d、9 d、12 d后ALP活性和首次处理12 h后BMP⁃2、Runx⁃2、OPG基因表达情况。结果6种强度的脉冲电磁场均促进细胞增殖,其中0�6mT的促增殖效应最强(P<0�01)。在成骨性诱导培养条件下,细胞内ALP活性随电磁场处理时间的延长而逐渐增加,第9 d达到峰值,之后开始回落。0�6 mT、3�0 mT和3�6 mT均能提高ALP活性,其中0�6 mT始终处于最高水平(P<0�01)。三种基因的表达水平在首次处理12h,在0�6mT、1�2mT、3mT和3�6mT组均显著提高,但仍然是0�6mT的增强效应最为明显。结论0�6mT50Hz脉冲电磁场具有最佳的促进MC3T3⁃E1细胞增殖和成骨性分化活性,这可能为采用脉冲电磁场治疗骨质疏松症提供了理想的治疗参数。%Objective To investigate the effect of pulsed electromagnetic fields ( PEMFs) on proliferation and differentiation of MC3T3⁃E1 cells. Methods MC3T3⁃E1 cells were divided into 7 groups after subculture. They were exposed to 50 Hz PEMFs (0�0, 0�6, 1�2, 1�8, 2�4, 3�0, and 3�6 mT, respectively) for 90 min/day. Cell proliferation was assessed using MTT method. ALP activity was examined on 3, 6, 9, and 12 days. Expression levels of BMP⁃2, Runx⁃2, and OPG mRNA were analyzed after 12 hours of PEMFs. Results Six intensities of PEMFs promoted MC3T3⁃E1 cell proliferation. The highest promotion was achieved under 0�6 mT intensity (P<0�01). Under the osteogenic culture condition, ALP activity gradually increased along with the time of exposure PEMFs. The peak was on the 9th day

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-10-01

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

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

    Science.gov (United States)

    Sinlapabodin, Salita; Amornsudthiwat, Phakdee; Damrongsakkul, Siriporn; Kanokpanont, Sorada

    2016-01-01

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

  4. Protective Effects of Statins on TNFa-induced Growth Inhibition in MC3T3-E1 Osteoblastic Cells%他汀类药物对TNFα诱导成骨细胞生长抑制的保护作用

    Institute of Scientific and Technical Information of China (English)

    赵奇江; 李芬芬; 董新威; 徐哲荣; 杨云梅; 谢强敏

    2012-01-01

    OBJECTIVE To study if four statins could attenuate TNFa-induced growth inhibition and compare their effects in MC3T3-E1 osteoblastic cells. METHODS Murine osteoblastic MC3T3-E1 cells were cultured in DMEM essential medium supplemented with 10% fetal bovine serum and antibiotics. 3-(4,5-Dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) was used to evaluate cell viability. RESULTS TNFa at concentrations (1-100 ng·mL‐1) inhibited the viability of MC3T3-E1 cells in time and concentration-dependent manners. MC3T3-E1 cells exposed to low concentrations of simvastatin(10‐10-10‐7mol·L‐1), fluvastatin(10‐10-10‐6mol·L‐1) and atorvastatin(l0‐8-10‐6mol·L‐1) increased growth and viability. In contrast, rosuvastatin(10‐10-10‐7 mol·L‐1) had no effect, and high concentration of rosuvastatin(10‐6-10‐5 mol·L‐1) decreased cell growth. MC3T3-E1 cells treated with simvastatin, fluvastatin and atorvastatin in the presence of TNFa concentration-dependently increased cell growth compared with that of untreated control cells. Low concentration of rosuvastatin(10‐l0-10‐8 mol·L‐1) in the presence of TNFa markedly increased cell growth, and high concentration of rosuvastatin(10‐6 mol·L‐1) in the presence of TNFa decreased cell growth. CONCLUSION The results demonstrate that statins can increase growth and viability of osteoblastic cells and decrease TNFa induced growth inhibition in murine osteoblastic MC3T3-E1 cells, and rosuvastatin at low and high concentration had opposite effects.%目的 比较4种他汀类药物对TNF-α诱导的小鼠成骨细胞(MC3T32-E1)生长抑制的影响.方法 小鼠成骨细胞MC3T3-E1用DMEM+10%胎牛血清培养,细胞活性用MTT法测定.结果 TNFα (1~100 ng·mL-1)呈浓度和时间依赖抑制MC3T3-E1细胞生长;低浓度的辛伐他汀(10-1010-7 mol·L-1)、氟伐他汀(10-10~10-6 mol·L-1)和阿托伐他汀(10-8~ 10-6 mol·L-1)处理 MC3T3-E1细胞72 h后能明显促进成骨细

  5. Tmed2 accelerates murine MC3T3-E1 pre-osteoblast proliferation in vitro%Tmed2促进体外小鼠前成骨细胞MC3T3-E1增殖

    Institute of Scientific and Technical Information of China (English)

    熊元; 熊霞辉; 卢雅彬; 朱宁; 陈梅红

    2012-01-01

    目的 研究Tmed2基因对小鼠前成骨细胞增殖的影响.方法1)分别在小鼠MC3T3-E1细胞中过表达和抑制Tmed2,检测细胞增殖情况.2)用雌激素处理细胞后,检测细胞的增殖及Tmed2基因的表达量.荧光实时定量PCR检测mRNA水平,MTS法检测细胞活力和增殖,流式细胞术检测细胞周期,Western blot法检测蛋白水平.结果 过表达Tmed2使MC3T3 -E1细胞的增殖速度加快,细胞周期中S期细胞比例明显增加,且Cyclin A的表达升高.而抑制Tmed2基因表达使MC3T3-E1细胞的增殖速度减慢.雌激素处理使细胞增殖速度加快的同时,Tmed2基因的表达显著增高.结论 Tmed2通过上调Cyclin A的表达,使S期细胞比例增加,加快小鼠前成骨细胞MC3T3-E1的增殖.此外,Tmed2的表达受雌激素的调控,可能参与雌激素促进MC3T3-E1细胞增殖的作用.%Objective To study the role of Tmed2 in murine pre-osteoblast proliferation. Methods 1) Over-express and knock down Tmed2, and the proliferation rate of MC3T3-E1 cell was then detected. 2) MC3T3-E1 cells were treated with p-Estradiol, cell number was then counted and the mRNA level of Tmed2 was analyzed. Quantitative real-time PCR was used to measure the mRNA level, MTS assay was used to assess the cell proliferation rate, flow eytometry was used to figure out the cell cycle distribution, and the protein level was evaluated by Western blot. Results Over-expression of Tmed2 accelerated MC3T3-E1 cell proliferation, and the proportion of cells in S phase markedly increased. The expression of Cyclin A also increased. On the contrary, Knockdown of Tmed2 decelerated MC3T3-E1 cell proliferation. In β-Estradiol promoted MC3T3-E1 proliferation, the mRNA level of Tmed2 remarkably increased. Conclusions Tmed2 accelerates murine pre-osteoblast MC3T3-E1 proliferation through up-regulating Cyclin A expression and therefore increasing the proportion of cells in S phase. In addition, the expression of Tmed2 is regulated by

  6. The effects of platelet-rich fibrin extract (PRFe) on osteoblast MC3T3-E1 cells%富血小板纤维蛋白提取液对成骨细胞影响的实验研究

    Institute of Scientific and Technical Information of China (English)

    董凯; 柳忠豪; 张晓洁; 许丰伟

    2013-01-01

    目的:探讨富血小板纤维蛋白提取液(PRFe)对成骨细胞增殖、分化及细胞骨架的影响.方法:将培养中的小鼠成骨细胞MC3T3-E1分为2组,实验组采用50%浓度PRFe,对照组为正常αt-MEM培养液.MTr法检测细胞增殖;碱性磷酸酶(ALP)试剂盒检测ALP活性;茜素红染色观察细胞矿化功能,并用图像分析软件进行半定量分析;激光共聚焦显微镜观察细胞骨架形态.结果:MTT实验显示随时间延长,细胞数目明显增加(P<0.05),在各时间点,实验组细胞数量明显高于对照组(P<0.05);ALP检测显示随时间延长,ALP活性明显增大(P<0.05),在各时间点,实验组A值均显著大于对照组(P<0.05);茜素红染色显示随时间延长,钙结节染色的积分吸光度值逐渐增大(P<0.05),每一时间点,实验组的钙结节积分吸光度值大于对照组(P<0.05);细胞骨架观察显示在各时间点,实验组细胞骨架较对照组更加伸展.结论:PRFe能促进MC3T3-E1细胞的增殖、分化,对细胞骨架的排列和伸展有促进作用.%Objective:To evaluate the effects of platelet-rich fibrin extract (PRFe) on the proliferation and differentiation of osteoblast MC3T3-E1 cells.Methods:MC3T3-E1 cells were cultured in 50% PRFe (test group) and normal α-MEM respectively (controlgroup).The proliferation,alkaline phosphatase (ALP) activity and mineralization were examined by MTT assay,ALP Kit and Alizarin red dye staining respectively; the F-actin cytoskeleton was observed by confocal laser scaning microscopy (CLSM).Results:PRFe treatment increased the proliferation(P < 0.05),ALP activity(P < 0.05),and calcium nodus formation of MC3T3-E1 cells(P <0.05) in a time-dependant manner.At each time point,filaments in PRFe treated cells were more well spread than those in the untreated.Conclusion:PRFe may stimulate the proliferation and differentiation of osteoblasts and can promote the spread of F-actin cytoskeleton.

  7. Behavior of MC3T3-E1 Osteoblast Cultured on Chitosan Modified with Polyvinylpyrrolidone

    Institute of Scientific and Technical Information of China (English)

    XI Jing; GAO Yuan; KONG Lijun; GONG Yandao; ZHAO Nanming; ZHANG Xiufang

    2005-01-01

    The physical and chemical properties of four kinds of modified chitosan materials made by blending chitosan with polyvinylpyrrolidone (PVP) were investigated. All four of these modified chitosan materials were hydrophilic with water contact angles ranging from 59° to 69°. Fourier transform-infrared spectra of the modified materials showed a new band at 1288 cm-1, implying formation of a surface physical interpenetrating network structure. Enzyme linked immunosorbent assay results indicated that much less fibronectin was adsorbed on the modified materials than on only chitosan. The viability of MC3T3-E1 osteoblasts cultured on the materials was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide assay. The results show that adding PVP10000 into the chitosan promotes adhesion of MC3T3-E1 osteoblasts on the modified materials, but has no effect on cell growth and proliferation; while adding PVP40000 reduces cell adhesion, growth, and proliferation. The results suggest that the increased hydrophilicity of the material surface does not always improve its biocompatibility, which will influence the selection and design of biomaterials.

  8. Recombinant protein of tissue inhibitor of metaIloproteinase-3 induces apoptosis of mouse MC3T3-E1 osteoblasts

    Institute of Scientific and Technical Information of China (English)

    袁凌青

    2006-01-01

    Objective To investigate the action of recombinantprotein of tissue inhibitor of metalloproteinase-3 (TIMP-3) on apoptosis of MC3T3-E1 osteoblasts. Methods Cell survival rate and apoptosis were measured by MTT and ELISA respectively. The expressions of Fas, FasL, Bel-2, Bax, caspase-3 , caspase-8, cytochrome c and phosphorylations of JNK, p38 and extracellular signalregulated kinase (ERK) 1/2 were analysed by Western blotting. Results TIMP-3 reduced survival rate of MC3T3-E1 cells and promoted apoptosis of MC3T3-E1

  9. Rapamycin/sodium hyaluronate binding on nano-hydroxyapatite coated titanium surface improves MC3T3-E1 osteogenesis

    Science.gov (United States)

    Liu, Chao; Dong, Jian Yong; Yue, Lin Lin; Liu, Shao Hua; Wan, Yi; Liu, Hong; Tan, Wan Ye; Guo, Qian Qian; Zhang, Dong

    2017-01-01

    Endosseous titanium (Ti) implant failure due to poor biocompatibility of implant surface remains a major problem for osseointegration. Improving the topography of Ti surface may enhance osseointegration, however, the mechanism remains unknown. To investigate the effect of modified Ti surface on osteogenesis, we loaded rapamycin (RA) onto nano-hydroxyapatite (HAp) coated Ti surface which was acid-etched, alkali-heated and HAp coated sequentially. Sodium hyaluronate (SH) was employed as an intermediate layer for the load of RA, and a steady release rate of RA was maintained. Cell vitality of MC3T3-E1 was assessed by MTT. Osteogenesis of MC3T3-E1 on this modified Ti surface was evaluated by alkaline phosphatase (ALP) activity, mineralization and related osteogenesis genes osteocalcin (OCN), osteopontin (OPN), Collagen-I and Runx2. The result revealed that RA/SH-loaded nano-HAp Ti surface was innocent for cell vitality and even more beneficial for cell osteogenesis in vitro. Furthermore, osteogenesis of MC3T3-E1 showed significant association with the mammalian target of rapamycin (mTOR) phosphorylation by RA, which required further study about the mechanism. The approach to this modified Ti surface presented in this paper has high research value for the development of Ti-based implant. PMID:28182765

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

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Koichi, E-mail: shimada-ki@dent.nihon-u.ac.jp [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo (Japan); Ikeda, Kyoko [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Ito, Koichi [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo (Japan)

    2009-12-18

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

  11. Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures.

    Science.gov (United States)

    Addison, William N; McKee, Marc D

    2010-04-01

    Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6 to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin gene expression.

  12. Effect of deferasirox on biological activity of murine preosteoblast MC3T3-E1 cells%地拉罗司对小鼠前成骨细胞MC3 T3-E1生物学活性的影响

    Institute of Scientific and Technical Information of China (English)

    赵国阳; 狄东华; 汪升; 徐又佳

    2016-01-01

    目的:探讨铁鳌合剂地拉罗司( deferasirox, DFS)体外对成骨细胞增生、分化、矿化和细胞内铁离子的影响。方法小鼠前成骨样细胞MC3T3-E1在37℃条件下体外培养,在10 mmol/L β-甘油磷酸和50 mg/L抗坏血酸的诱导作用下,分化为成骨细胞,同时用不同浓度(10、20、40μmol/L ) DFS 干预,用CCK-8法检测细胞的增生,碱性磷酸酶( alkaline phosphatase, ALP)活性试剂盒检测细胞ALP活性, Von kos-sa染色法行细胞钙结节染色,实时定量PCR检测细胞膜转铁蛋白受体( transferrin receptor, TfR) mRNA的表达。结果 MC3T3-E1细胞增生结果显示, DMSO溶剂组、对照组(0μmol/L)、10、20、40μmol/L组A值分别为1.41±0.09、1.41±0.09、1.01±0.01、0.79±0.04、0.67±0.04; ALP 活性检测结果显示,对照组(0μmol/L)、10、20、40μmol/L组ALP活性值分别为0.73±0.03、0.65±0.02、0.54±0.03、0.35±0.04;钙结节检测结果显示,对照组(0μmol/L)、10、20、40μmol/L组TfR mRNA钙化面积比值分别为4.22±0.12、3.29±0.14、1.40±0.20、0.86±0.21; TfR mRNA表达检测结果显示,对照组(0μmol/L)、10、20、40μmol/L组TfR mRNA表达比分别为1、1.52±0.23、1.91±0.17、2.98±0.14。 MC3T3-E1细胞的增生、ALP活性、钙结节和钙化面积随DFS干预浓度的增加呈剂量依赖性降低( P<0.05), TfR mRNA的表达随DFS干预浓度的增加呈剂量依赖性升高(P<0.05)。结论 DFS可能通过螯合成骨细胞内的铁离子抑制其增生、分化和矿化。%Objective To investigate the effects of deferasirox ( DFS) on proliferation, differentiation, miner-alization and intracellular iron of osteoblast in vitro.Methods Murine preosteoblast MC3T3-E1 cells were incubated in a medium supplemented with different concentrations (10, 20, 40 μmol/L) of

  13. Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures.

    Science.gov (United States)

    Hoac, Betty; Kiffer-Moreira, Tina; Millán, José Luis; McKee, Marc D

    2013-04-01

    Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and β-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10μM. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as

  14. An analysis on fluorine-induced autophagy and apoptosis of MC3T3-E1 and their interaction%氟诱导成骨细胞MC3T3-E1自噬与凋亡及其相互作用分析

    Institute of Scientific and Technical Information of China (English)

    郭晓东; 杨茂伟; 梁单; 郭宝磊; 杨蕾; 曹军军

    2012-01-01

    目的 主要研究氟诱导成骨细胞MC3T3-E1发生自噬、凋亡及两者之间的关系.方法 利用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法检测氟对成骨细胞MC3T3-E1的增殖活性影响,探寻氟引起凋亡的浓度;流式细胞术检测细胞凋亡;western blot免疫印记技术检测凋亡相蛋白;利用western blot免疫印记技术检测Beclin 1蛋白及LC3蛋白表达水平及变化.结果 氟能明显抑制成骨细胞MC3T3-E1增殖,诱发细胞凋亡,其抑制作用呈时间、剂量依赖性;氟在促进细胞凋亡的同时也促进自噬产生;3-甲基腺素(3-MA)抑制自噬后,氟诱导的凋亡进一步增多.结论 氟明显抑制MC3T3-E1细胞增殖,同时诱发细胞凋亡和自噬;氟诱导的自噬部分拮抗其诱导的凋亡.%Objectives; To explore Fluorine - induced apoptosis and autophagy of MC3T3 - E1 cells and the relationship between them. Methods To explore the concentration of fluorine that caused cell apoptosis the influence of fluorine on proliferative vitality of MC3T3 - E1 was assessed by MTT assay. The apoptosis of MC3T3 - E1 was delected by flow cytometry;The expression of apoptosic protein was determined by Western Blot ;The LC3 and the Beclinl protein level and variation were analyzed by west-em blot assay. Results Fluorine inhibited the proliferation of MC3T3 - E1 cells dose and time dependency and simultaneously induced apoptosis and autophagy; Inhibition of autophagy by 3 - MA promotes fluorine - induced apoptosis. Conclusion Fluorine can inhibit cell proliferation and induce apoptosis and autophagy, but autophagy partly atagonizes apoptosis.

  15. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

  16. TiO2纳米管对MC3T3-E1前成骨细胞骨功能基因表达变化的影响%MC3T3-E1 Preosteobalst Osteogenic Gene Expression on Titanium with the Nanotube Layer.

    Institute of Scientific and Technical Information of China (English)

    于卫强; 徐玲; 张富强

    2011-01-01

    目的:研究阳极氧化TiO2纳米管对MC3T3-E1前成骨细胞骨功能基因表达变化的影响.方法:采用阳极氧化法在钛基底表面制备TiO2纳米管,扫描电镜观察其表面微结构,肌动蛋白染色研究细胞在材料表面的粘附生长情况,Real-time PCR技术系统分析材料对细胞骨功能基因表达的影响规律.结果:MC3T3-E1前成骨细胞在TiO2纳米管表面比光滑钛有更大的伸展面积,培养1周后,TiO2纳米管提高了细胞骨功能基因碱性磷酸酶、骨桥蛋白和骨钙蛋白的表达水平(P<0.05),培养2周后,骨功能基因表达水平与对照组之间没统计学差异.结论:TiO2纳米管可以促进MC3T3-E1前成骨细胞肌动蛋白组装,同时可以提高细胞骨功能基因的早期表达水平.%Objective: To evaluate the MC3T3-E1 preosteobalst osteogenic gene expression on the TiO2 nanotube layer in vitro. Methods: The TiO2 nanotube layer was grown by anodization. SEM experiment method has been employed to characterize the nanotubes. The action cytoskeletons of cells were visualized by a confocal laser scanning microscopy. Real-time PCR has been used to evalute bone gene expression of preosteoblast. Results.. The area of preosteoblast cultured on TiO2 nanotube layer was larger than that on smooth titanium. The nanotubes could promote the ALP, OPN, and OCN gene expression after one week of cultivation by analysing real-time PCR results (P<0. 05), but not after two weeks of cultivation. Conclution: Actin cytoskeleton of cells cultured on the nanotubes shows more arrangement than the smooth Ti. The TiO2 nanotube layer can promote bone gene expression at earlier period.

  17. mTORC1 signaling promotes osteoblast differentiation of preosteoblast MC3 T3-E1%mTORC1信号促进前成骨细胞MC3 T3-E1的分化成熟

    Institute of Scientific and Technical Information of China (English)

    张婧婧; 张楠; 吴伟; 王鹏; 杨景瑞; 段巨洪; 于海川

    2016-01-01

    目的:研究mTORC1信号对前成骨细胞MC3T3-E1向成骨细胞分化成熟的调控作用。方法:通过向MC3T3-E1转染pcDNA3.1-Raptor,对mTORC1信号相关蛋白Raptor进行过表达。向MC3T3-E1转染Raptor siRNA,对mTORC1信号蛋白Raptor进行基因沉默。通过Real-time PCR方法测定Raptor的基因表达,通过蛋白免疫印迹法测定Raptor蛋白水平,并通过茜素红染色检测成骨矿化情况,以测定成骨分化程度。通过Real-time PCR检测成骨分化指标的基因表达。结果:与对照组相比,Raptor过表达组的Raptor mRNA和蛋白水平明显增加;茜素红染色结果显示Raptor过表达组染色更深,说明成骨矿化程度更高;荧光定量PCR结果显示,Raptor过表达组的成骨分化标记基因以及成骨转录因子的表达量均高于对照组。与对照组相比,Raptor siRNA组的Raptor mRNA和蛋白水平明显降低;茜素红染色结果显示Raptor siRNA组染色更浅,说明成骨矿化程度更低;荧光定量PCR结果显示,Raptor siRNA组的成骨分化标记基因以及成骨转录因子的表达量均低于对照组。结论:mTORC1信号促进前成骨细胞MC3T3-E1向成骨细胞分化成熟。%Objective:To investigate the regulatory role of mTORC 1 signaling on osteoblast differentiation from preosteoblast cell MC3T3-E1.Methods:To overexpress the gene Raptor ,which was a critical component of mTORC1 signaling,pcDNA3.1-Raptor was transfected to preosteoblast cell MC 3T3-E1.The Raptor overexpression was confirmed by Real-time PCR and Western blot.For the gene silence of Raptor ,Raptor siRNA was transfected to preosteoblast cell MC 3T3-E1.Alizarin red staining was used to detect the min-eralization of MC3T3-E1.Real-time PCR was used to detect the gene expression of osteoblast specific marker genes.Western blot was used to detect the protein level of Raptor.Results: Compared to the control group ,the levels of Raptor mRNA and proteins increased

  18. Riboflavin and photoproducts in MC3T3-E1 differentiation

    NARCIS (Netherlands)

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

    2010-01-01

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

  19. The New Synthetic H2S-Releasing SDSS Protects MC3T3-E1 Osteoblasts against H2O2-Induced Apoptosis by Suppressing Oxidative Stress, Inhibiting MAPKs, and Activating the PI3K/Akt Pathway.

    Science.gov (United States)

    Yan, Xiaofei; Wu, Haixia; Wu, Zhiyuan; Hua, Fei; Liang, Dong; Sun, Hong; Yang, Yong; Huang, Dejian; Bian, Jin-Song

    2017-01-01

    Reactive oxygen species (ROS) are important in osteoporosis development. Oxidative stress induces apoptosis of osteoblasts and arrest of their differentiation. Both Danshensu (DSS) and hydrogen sulfide (H2S) produce significant antioxidant effect in various systems. In this study, we synthesized SDSS, a novel H2S-releasing compound derived from DSS, and studied its antioxidant effect in an H2O2-induced MC3T3-E1 osteoblastic cell injury model. We first characterized the H2S releasing property of SDSS in both in vivo and in vitro models. HPLC chromatogram showed that intravenous injection of SDSS in adult rats released ADT-OH, a well proved H2S sustained-release moiety, within several minutes in the rat plasma. Using an H2S selective fluorescent probe, we further confirmed that SDSS released H2S in MC3T3-E1 osteoblastic cells. Biological studies revealed that SDSS had no significant toxic effect but produced protective effects against H2O2-induced MC3T3-E1 cell apoptosis. SDSS also reversed the arrest of cell differentiation caused by H2O2 treatment. This was caused by the stimulatory effect of SDSS on bone sialoprotein, runt-related transcription factor 2, collagen expression, alkaline phosphatase activity, and bone nodule formation. Further studies revealed that SDSS reversed the reduced superoxide dismutase activity and glutathione content, and the increased ROS production in H2O2 treated cells. In addition, SDSS significantly attenuated H2O2-induced activation of p38-, ERK1/2-, and JNK-MAPKs. SDSS also stimulated phosphatidylinositol 3-kinase/Akt signaling pathway. Blockade of this pathway attenuated the cytoprotective effect of SDSS. In conclusion, SDSS protects MC3T3-E1 cells against H2O2-induced apoptosis by suppressing oxidative stress, inhibiting MAPKs, and activating the phosphatidylinositol 3-kinase/Akt pathway.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-12-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  2. Effects of Quenching Temperature and Time on Pore Diameter of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Porous Scaffolds and MC3T3-E1 Osteoblast Response to the Scaffolds

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds were prepared by thermally inducing phase separation (TIPS) for bone reconstruction. Scanning electron microscopy and porosity measurements were used to analyze the structure and properties of the scaffolds. The pore diameter of the scaffolds could be easily controlled by changing the quenching temperature and time. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 osteoprogenitor cells seeded on the scaffolds. Cultures grown in the presence of a source of phosphate ions showed the formation of a mineralized extracellular matrix. The results indicate that PHBHHx scaffolds prepared using TIPS are a promising candidate for bone reconstruction.

  3. The roles of PI3K/Akt signaling pathway in regulating MC3T3-E1 preosteoblast proliferation and differentiation on SLA and SLActive titanium surfaces.

    Science.gov (United States)

    Gu, Ying-Xin; Du, Juan; Si, Mi-Si; Mo, Jia-Ji; Qiao, Shi-Chong; Lai, Hong-Chang

    2013-03-01

    Chemical modification to produce a hydrophilic microrough titanium (Ti) implant surface has been shown to increase osseointegration compared with microrough topography alone. This study aimed to investigate the roles of PI3K/Akt signaling pathway in regulating proliferation and differentiation of osteoblasts in response to surface microroughness and hydrophilicity. Ti disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT), a rough hydrophobic surface that was sand-blasted, large-grit, acid-etched (SLA), and an SLA surface with the same roughness that was chemically modified to possess high wettability/hydrophilicity (SLActive/modSLA). MC3T3-E1 cells were cultured on these substrates with or without LY294002, a PI3K inhibitor, and their behaviors, including cell viability (MTT colorimetric assay), alkaline phosphatase (ALP) activity, and osteogenic genes expression of osteopontin (OPN) and osteocalcin (OCN) were measured. Western blot was applied to detect the expression of PI3K/Akt signal pathway proteins. The results showed that a decrease in osteoblast proliferation associated with the Ti surfaces (SLActive > SLA > PT) correlated with an increase in activity of the osteogenic differentiation markers ALP. The peak of ALP activity appeared earlier at 7 days for the SLActive surfaces compared with the SLA and PT surfaces. Osteoblast proliferation, as well as the level of p-Akt, was significantly inhibited by LY294002 in all three Ti surfaces. The top value of ALP activity was increased with the inhibition of PI3K/Akt signaling pathway while the time of the peak appeared was not advanced. The expression levels of OPN and OCN were upregulated by the effect of surface roughness and hydrophilicity, which were further enhanced by LY294002. In conclusion, osteogenic responses to SLActive surface were moderately better than the SLA surface and protein expression studies indicated that PI3K/Akt signaling activation may be

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

    NARCIS (Netherlands)

    Wang, Jiawei; Boer, de Jan; Groot, de Klaas

    2008-01-01

    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 prolife

  5. 0.5 Gy X线照射对MC3T3-E1成骨细胞基因表达谱的影响%Effects of 0.5 Gy X-ray radiation on the profile of gene expression in MC3T3-E1 osteoblasts

    Institute of Scientific and Technical Information of China (English)

    陈明; 董启榕; 黄群; 徐炜; 佘昶

    2016-01-01

    Objective To investigate the molecular mechanism of low-dose X-ray irradiation on osteoblasts by detecting the gene expression profiles of osteoblasts radiated with 0.5 Gy X-ray.Methods Microarray analyses to value the changes of gene expression of MC3T3-E1 osteoblasts after 0.5 Gy X-ray irradiation were conducted.The end points included modulation of key markers,and pathway and gene ontology through transcriptomic profiling and bioinformatics analysis.Several major genes during osteoblasts differentiation were selected for Real-time PCR analysis.Results 1 412 differentially expressed transcripts were identified between the radiated group and control group.Among the identified genes,559 transcripts were up-regulated and 853 transcripts were down-regulated after irradiation.Real-time PCR analysis indicated that the mRNA expressions of Bglap-rs1,Col1a2,Tgf-β1,Lrp5,Dvl1,Map4k5 were apparently higher than that of the controls (P < 0.01),whereas the Id2 and Dkk1 expression decreased under the same condition (P < 0.01).Western-blot analysis indicated TGF-β1 and Lrp-5 protein expression increased and Dkk1 expression decreased(P < 0.05).Pathway analysis and gene ontology analysis revealed that focal adhesion,extracellular signaling,cytoskeletal protein binding,Wnt signal pathway,IκB/NF-κB signal pathway and growth factor receptors activities.Conclusions Low-dose X-ray radiation promoted osteoblasts differentiation in which focal adhesion,extracellular signaling,cytoskeletal protein binding,Wnt signal pathway,IκB/NF-κB signal pathway and growth factor activities might be involved.%目的 探索低剂量X线促进成骨分化的关分子机制.方法 0.5 Gy X线照射成骨细胞后继续诱导成骨培养,采用基因芯片技术检测X线照射成骨细胞后基因表达谱的变化,筛检出表达差异基因,选取部分骨分化相关基因通过实时聚合酶链反应(real-time PCR)进行验证,并进行基因本体(Gene Ontology,GO)

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

    Directory of Open Access Journals (Sweden)

    Kathryn Dorst

    2014-02-01

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

  7. 富血小板纤维蛋白提取液对MC3T3-E1细胞影响的实验研究%Effects of platelet-rich fibrin extract on MC3T3-E1 cell

    Institute of Scientific and Technical Information of China (English)

    董凯; 柳忠豪; 张晓洁; 许丰伟

    2013-01-01

    Objective To evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts.Methods The experimental group used the α-minimum essential medium(α-MEM) containing PRFe(10% fetal bovine serum),and the control group used the α-MEM (10% fetal bovine serum).The number of the osteoblasts at 1st,3rd,5th d was detected by methyl thiazolyl tetrazolium(MTT) assay,and the differentiation of osteoblast at lst,3rd,5th,7 th d detected by the activity of alkaline phosphatase(ALP).The alizarin red dye was used to observe the number of calcium nodus at 14th,21st d.The F-actin cytoskeleton was evaluated by confocal laser scanning microscope (CLSM) at 3rd,6th,9th,12th h.The level of osteogenetic biomarkers osteocalcin(OCN) and core-binding factor α1 (Cbfα1) at 3rd,7th d were quantified by real-time PCR.Results A significant increase of absorbance at 1st,3rd,5th d was showed in experimental group (0.336 ± 0.011,0.571 ± 0.039,0.787 ± 0.050) compared to control group (0.300 ± 0.021,0.387 ±0.040,0.527 ±0.034) (P <0.05).The absorbance of experimental group at lst,3rd,5th,7th d(0.146 ± 0.014,0.199 ±0.017,0.390 ±0.020,0.492 ±0.019) was significantly higher than that of control group(0.115 ± 0.014,0.145 ± 0.015,0.190 ± 0.015,0.230 ± 0.026) (P < 0.05).The integrated absorbance of the calcium nodus in experimental group at 14th,21st d (22.119 ± 3.694,31.528 ± 3.162) was significantly higher than in control group(8.498 ±2.041,15.162 ±2.526) (P <0.05).The Cbfα1 and OCN gene expression in experimental group was higher than in control group (P < 0.05).Conclusions PRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin cytoskeleton.%目的 探讨富血小板纤维蛋白提取液(platelet-rich fibrin extract,PRFe)对成骨细胞增殖、分化及细胞骨架的影响,以期为富血小板纤维蛋白在临床的应用提供理论基础.方法 实验组使用含PRFe的α-最低必需培养基(α-minimum essential medium,α-MEM)培养液(10%胎牛血清)培养细胞,对照组使用不含PRFe的α-MEM培养液(10%胎牛血清).甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法检测培养1、3、5d细胞增殖的A值;以碱性磷酸酶(alkaline phosphatase,ALP)活性检测1、3、5、7d的细胞分化情况;茜素红染色观察14、21 d细胞分泌钙结节的情况;激光共聚焦显微镜观察培养后3、6、9及12 h的细胞骨架形态;荧光实时定量PCR检测核心结合因子α1(core-binding factor α1,Cbfα1)和骨钙蛋白基因分别在培养3、7d的表达.结果 MTT检测结果显示,随培养时间延长,细胞数量逐渐增加,在培养1、3、5d实验组细胞A值(分别为0.336±0.011、0.571±0.039、0.787±0.050)均显著高于对照组(分别为0.300±0.021、0.387±0.040、0.527±0.034) (P <0.05).两组ALP活性均随培养时间延长而增加,实验组的增加更为显著,在培养1、3、5、7d实验组A值(分别为0.146 ±0.014、0.199±0.017、0.390±0.020及0.492 ±0.019)均显著大于对照组(分别为0.115±0.014、0.145±0.015、0.190 ±0.015及0.230±0.026)(P<0.05).茜素红染色结果显示,两组随培养时间延长细胞分泌的钙结节均逐渐增多,培养14、21 d实验组细胞分泌的钙结节积分吸光度值(分别为22.119±3.694、31.528±3.162)均较对照组(分别为8.498±2.041、15.162 ±2.526)显著增加(P<0.05).在各时间点,实验组细胞骨架均较对照组更加伸展;两组细胞的基因表达量也随时问逐渐增加,实验组细胞Cbfα1和骨钙蛋白基因表达量均显著高于对照组(P<0.05).结论 PRFe能有效促进成骨细胞的增殖和分化,并对细胞骨架早期的排列和伸展有明显的促进作用.

  8. Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells.

    Science.gov (United States)

    Gale, Zoe; Cooper, Paul R; Scheven, Ben A

    2012-02-01

    Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.

  9. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    Energy Technology Data Exchange (ETDEWEB)

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  10. Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

    Science.gov (United States)

    Cobo, Teresa; Viloria, Cristina G.; Solares, Laura; Fontanil, Tania; González-Chamorro, Elena; De Carlos, Félix; Cobo, Juan; Cal, Santiago; Obaya, Alvaro J.

    2016-01-01

    Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology. PMID:26809067

  11. Experiment list: SRX471336 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available : MC3T3-E1 cells day28 DNase-seq; Mus musculus; DNase-Hypersensitivity source_name=mineralizing MC3T3-E1 sub...clone 4 cells at day 28 || cell line=MC3T3-E1 || time=day28 || cell type=mineralizing osteoblast || passages

  12. Chitosan and polycaprolactone membranes patterned via electrospinning: effect of underlying chemistry and pattern characteristics on epithelial/fibroblastic cell behavior.

    Science.gov (United States)

    Simşek, Murat; Capkın, Merve; Karakeçili, Ayşe; Gümüşderelioğlu, Menemşe

    2012-12-01

    Electrospinning was used as an effective route to pattern chitosan (CS) and polycaprolactone (PCL) membranes with submicron fibers having different chemical structure (PCL or PCL/collagen) and physical characteristics (size: between ≈200 and 550 nm; randomly oriented or aligned form). While the PCL fibers with diameters in the same range (≈200 nm) were patterned on both of CS and PCL membranes to evaluate the influence of the underlying membrane chemistry, only CS membranes were patterned with PCL fibers having different sizes simply by changing the electrospinning conditions to investigate the effects of pattern characteristics. Furthermore, collagen was added to the PCL fiber structure to change the chemical composition of the fibers in a cell-attractive way. Two cell lines with different morphologies, fibroblastic MC3T3-E1 preosteoblasts and epithelial Madine Darby Bovine Kidney (MDBK) cells, were cultured on the patterned membranes. The observation of cellular behavior in terms of cell morphology and F-actin synthesis was realized by scanning electron microscopy and confocal microscopy analysis during the first 12 h of culture period. The viability of cells was controlled by MTT assay through 96 h of cell culture. The cell culture studies indicated that the leading aspect for the morphology change on patterned membranes was the fiber orientation. The aligned topography controlled the morphology of cells both on CS and PCL membranes. In the presence of collagen in the fiber structure, F-actin filament synthesis increased for MC3T3-E1 and MDBK cell lines.

  13. Uptake of nickel from 316L stainless steel into contacting osteoblastic cells and metal ion interference with BMP-2-induced alkaline phosphatase.

    Science.gov (United States)

    Mölders, Martina; Felix, Joachim; Bingmann, Dieter; Hirner, Alfred; Wiemann, Martin

    2007-11-01

    Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.

  14. Morphogenetic study on the maturation of osteoblastic cell as induced by inorganic polyphosphate.

    Directory of Open Access Journals (Sweden)

    Kaori Tsutsumi

    Full Text Available Since inorganic polyphosphates [poly(P] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P. Ultrastructural analysis of cells cultured with poly(P revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P, it implies that poly(P can be an effective material for bone regeneration.

  15. Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

    Science.gov (United States)

    Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

    Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

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

    Energy Technology Data Exchange (ETDEWEB)

    Kongsfelt, Iben Boutrup [Department of Molecular Biology and Genetics, Aarhus University, Aarhus (Denmark); Byskov, Kristina [Department of Molecular Biology and Genetics, Aarhus University, Aarhus (Denmark); Department of Clinical Medicine, Aarhus University, Aarhus (Denmark); Pedersen, Lasse Ebdrup [Department of Molecular Biology and Genetics, Aarhus University, Aarhus (Denmark); Pedersen, Lene, E-mail: LP@mb.au.dk [Department of Molecular Biology and Genetics, Aarhus University, Aarhus (Denmark); Department of Clinical Medicine, Aarhus University, Aarhus (Denmark); Department of Hematology, Aarhus University Hospital, Aarhus (Denmark)

    2014-08-01

    The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M within 12 h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24 h after seeding. - Highlights: • Effects of elevated levels of the inorganic phosphate transporter PiT1 were studied. • The density-inhibited murine cell lines NIH3T3 and MC3T3-E1 showed faster adhesion. • NIH3T3 cells showed faster spreading. • We suggest that the faster adhesion/spreading contributes to faster proliferation.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-11-01

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

  18. Regulation of cell proliferation and cell density by the inorganic phosphate transporter PiT1

    Directory of Open Access Journals (Sweden)

    Byskov Kristina

    2012-03-01

    Full Text Available Abstact Background The inorganic phosphate (Pi transporter, PiT1 (SLC20A1, is ubiquitously expressed in mammalian cells. It has previously been shown that down-regulation of PiT1 severely impaired the proliferation of two transformed human cells lines, HepG2 and HeLa, and the tumorigenicity of HeLa cells in nude mice. Moreover, PiT1 knock-out mice do not survive past E12.5 and from E10.5, the embryos were found to be growth-retarded and showed reduced proliferation of liver cells. Isolated mouse embryonic fibroblasts with knocked out as well as reduced PiT1 expression levels also exhibited impaired proliferation. Together these results suggest that a certain level of PiT1 is important for proliferation. We have here investigated the role of PiT1 in regulation of cell proliferation using two strictly density-inhibited cells lines, the murine MC3T3-E1 and NIH3T3 cells. Results We found that knock-down of PiT1 in MC3T3-E1 cells led to impaired proliferation supporting that at least a certain level of PiT1 is important for wildtype level of proliferation. We, however, also observed that MC3T3-E1 and NIH3T3 cells themselves regulate their endogenous PiT1 mRNA levels with lower levels in general correlating with decreased proliferation/increased cell density. Moreover, over-expression of human PiT1 led to increased proliferation of both MC3T3-E1 and NIH3T3 cultures and resulted in higher cell densities in cultures of these two strictly density-inhibited cell lines. In addition, when we transformed NIH3T3 cells by cultivation in fetal bovine serum, cells over-expressing human PiT1 formed more colonies in soft agar than control cells. Conclusions We conclude that not only is a certain level of PiT1 necessary for normal cell division as suggested by previously published studies, rather the cellular PiT1 level is involved in regulating cell proliferation and cell density and an increased PiT1 expression can indeed make NIH3T3 cells more sensitive to

  19. CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

    Science.gov (United States)

    Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

    2016-08-01

    Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1.

  20. A 1-min method for homogenous cell seeding in porous scaffolds

    NARCIS (Netherlands)

    Tan, Lijun; Ren, Yijin; Kuijer, Roel

    2012-01-01

    The aim of this study was to develop and evaluate a simple and rapid cell seeding procedure for both calcium phosphate ceramic scaffolds and polymer scaffolds. Poly(D,L-lactic acid) and beta-tri-calcium phosphate scaffolds were seeded with MC3T3-E1 cells in a syringe. Scaffolds were put in the syrin

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-03-11

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

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

    Science.gov (United States)

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

    2011-03-01

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

  3. Effect of contact with titanium alloys on the proliferation of mouse osteoblastic cells in culture.

    Science.gov (United States)

    Onuki, Hiroyuki; Sakagami, Hiroshi; Kobayashi, Masahiko; Hibino, Yasushi; Yokote, Yoshiko; Nakajima, Hiroshi; Shimada, Jun

    2010-01-01

    This study was aimed at studying the effect of contact with titanium alloy plates of different surface textures on the proliferative capability of mouse osteoblastic MC3T3-E1 cells. First, the proliferation characteristics of MC3T3-E1 cells were investigated. MC3T3-E1 cells showed a high capacity for proliferation and survived for a long period even under nutritionally starved conditions. During logarithmic cell growth, the consumption of Ser, Gln, Val, Ile and Leu increased time-dependently. Contact with an hydoxyapatite (HA)-coated titanium alloy plate resulted in the increase in the recovery of cells from the plate by trypsin, and an increase in the consumption of these amino acids, suggesting enhanced cell proliferation. On the contrary, contact with the sandblasted and anodized titanium alloy plates resulted in the reduction of the recovery of the cells from the plate, but a slight increase in the amino acid consumption, suggesting the tight adhesion of the cells to the plates. This study demonstrates that the present method, based on the amino acid consumption of the cells, is useful for monitoring the cell proliferative capability, without detachment of the cells from the plate. This method may be applicable to the study of the interaction between cells and metal plates.

  4. Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

    Institute of Scientific and Technical Information of China (English)

    YUAN Ye; Wu Zhi-jun; YAO Hui-yu; YU Xiao-dan; GUO Zi-kuan; CHEN Xiao-san; TANG Pei-xian; MAO Ning

    2006-01-01

    @@ Bone morphogenetic proteins (BMPs) induce ectopic bone formation and promote osteoblast differentiation.1 It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad (Smad1, 5, 8) could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription.2

  5. Mineralization and Osteoblast Cells Response of Nanograde Pearl Powders

    Directory of Open Access Journals (Sweden)

    Jian-Chih Chen

    2013-01-01

    Full Text Available The main objective of this study is to characterize the thermal, mineralization, and osteoblast cells response of pearl nanocrystallites. The results obtained from X-ray diffraction, FTIR spectra demonstrate that the pearl nano-crystallites can induce the formation of an HA layer on their surface in SBF, even after only short soaking periods. The in vitro cell response to nano-grade pearl powders is assessed by evaluating the cytotoxicity against a mouse embryonic fibroblast cell line and by characterizing the attachment ability and alkaline phosphatase activity of mouse bone cells (MC3T3-E1, abbreviated to E1 and bone marrow stromal precursor (D1 cells. The cytotoxicities of pearls were tested by the filtration and culture of NIH-3T3 mouse embryonic fibroblast cells. The viability of the cultured cells in media containing pearl crystallites for 24 and 72 h is greater than 90%. The bone cells seen in these micrographs are elongated and align predominately along the pearl specimen. The cells observed in these images also appeared well attached and cover the surface almost completely after 1 h. The pearl nanocrystallites had a positive effect on the osteogenic ability of ALP activity, and this promoted the osteogenic differentiation of MSCs significantly at explanations.

  6. Famotidine suppresses osteogenic differentiation of tendon cells in vitro and pathological calcification of tendon in vivo.

    Science.gov (United States)

    Yamamoto, Kenichi; Hojo, Hironori; Koshima, Isao; Chung, Ung-il; Ohba, Shinsuke

    2012-12-01

    Heterotopic ossification or calcification follows any type of musculoskeletal trauma and is known to occur after arthroplasties of hip, knee, shoulder, or elbow; fractures; joint dislocations; or tendon ruptures. Histamine receptor H2 (Hrh2) has been shown to be effective for reducing pain and decreasing calcification in patients with calcifying tendinitis, which suggested that H2 blockers were effective for the treatment of tendon ossification or calcification. However, the detailed mechanisms of its action on tendon remain to be clarified. We investigated the mechanisms underlying H2 blocker-mediated suppression of tendon calcification, with a focus on the direct action of the drug on tendon cells. Famotidine treatment suppressed the mRNA expressions of Col10a1 and osteocalcin, ossification markers, in a tendon-derived cell line TT-D6, as well as a preosteoblastic one MC3T3-E1. Both of the cell lines expressed Hrh2; histamine treatment induced osteocalcin expression in these cells. Famotidine administration suppressed calcification in the Achilles tendon of ttw mice, a mouse model of ectopic ossification. These data suggest that famotidine inhibits osteogenic differentiation of tendon cells in vitro, and this inhibition may underlie the anti-calcification effects of the drug in vivo. This study points to the use of H2 blockers as a promising strategy for treating heterotopic ossification or calcification in tendon, and provides evidence in support of the clinical use of famotidine.

  7. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    Science.gov (United States)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX

  8. Cytocompatibility of brushite and monetite cell culture scaffolds made by three-dimensional powder printing.

    Science.gov (United States)

    Klammert, U; Reuther, T; Jahn, C; Kraski, B; Kübler, A C; Gbureck, U

    2009-02-01

    This study investigated the cytocompatibility of low-temperature direct 3-D printed calcium phosphate scaffolds in vitro. The fabrication of the scaffolds was performed with a commercial 3-D powder printing system. Diluted phosphoric acid was printed into tricalcium phosphate powder, leading to the formation of dicalcium phosphate dihydrate (brushite). Hydrothermal conversion of the brushite matrices led to the formation of dicalcium phosphate anhydrous (monetite). The biocompatibility was investigated using the osteoblastic cell line MC3T3-E1. Cell viability and the expression of alkaline phosphatase served as parameters. The culture medium was analyzed for pH value, concentration of free calcium and phosphate ions and osteocalcin. Both types of scaffolds showed a considerable increase of cell proliferation and viability; the monetite matrices were a little inferior compared with the brushite ones. The activity of alkaline phosphatase showed a similar pattern. Optical and electron microscopy revealed an obvious cell growth on the surface of both materials. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium and phosphate ions were obviously different among brushite and monetite cultures, due to their different solubility. The content of osteocalcin of the culture medium was reduced by the printed scaffolds due to adsorption. We conclude that the powder printed brushite and monetite matrices have a suitable biocompatibility for their use as cell culture scaffolds. Both materials enable osteoblastic cells in vitro to proliferate and differentiate due to the expression of typical osteoblastic markers.

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

    Directory of Open Access Journals (Sweden)

    Barrio Daniel A

    2001-08-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    of time with regard to the two-dimensional pattern and vertical dimension of the structure. Microstructures of parallel grooves/ridges caused elongated cell growth after 1 and 4 h in comparison to a flat, nonstructured, reference surface. For microstructures consisting of pillars, cell spreading was found......, and length of preosteoblastic cells (MC3T3-E1). Cells were examined after 0.5, 1, 4, and 24 h on different Ta microstructures with vertical dimensions (heights) of 0.25 and 1.6 mu m. Cell morphologies depended upon the underlying Surface topography, and the length and spreading of cells varied as a function...

  11. Osteoblast-conditioned media influence the expression of E-selectin on bone-derived vascular endothelial cells.

    Science.gov (United States)

    Makuch, Lauren A; Sosnoski, Donna M; Gay, Carol V

    2006-08-01

    Breast cancer cells frequently metastasize to the ends of long bones, ribs and vertebrae, structures which contain a rich microvasculature that is closely juxtaposed to metabolically active trabecular bone surfaces. This study focuses on the effects of osteoblast secretions on the surface presentation of adhesive proteins on skeletal vascular endothelial cells. Vascular endothelial cells were isolated from trabecular bone regions of the long bones of 7-week-old Swiss Webster mice and also from the central marrow cavity where trabecular bone is absent. Both types of endothelial cells were placed in culture for 7 days, then exposed 24 h to conditioned media from MC3T3-E1 osteoblasts. Conditioned medium (CM) from two different stages of osteoblast development were tested: (1) from immature MC3T3-E1 cells cultured for 5-7 days and (2) from mature MC3T3-E1 cells cultured for 28-30 days. The immature osteoblasts were in a stage of rapid proliferation; the mature osteoblasts formed a matrix that mineralized. Following exposure to the conditioned media, the vascular cells were exposed to anti-P-selectin, anti-E-selectin, anti-ICAM-1, and anti-VCAM-1 to detect the corresponding adhesive proteins on their surfaces. Breast cancer cells are known to bind to these adhesive proteins. Of the four proteins evaluated, E-selectin was consistently found on more cell surfaces (approximately 30%) of bone-derived vascular endothelial cells (BVECs) when exposed to the immature CM whereas vascular endothelial cells from marrow (MVECs) did not show this response to either immature CM or mature CM. These studies suggest that the BVEC blood vessels near immature bone cells express more surface adhesive protein that could enhance entrapment and extravasation of breast cancer cells. Once cancer cells have undergone extravasation into marrow adjacent to bone, they could be readily attracted to nearby bone surfaces.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-15

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

  13. One-day treatment of small molecule 8-bromo-cyclic AMP analogue induces cell-based VEGF production for in vitro angiogenesis and osteoblastic differentiation.

    Science.gov (United States)

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

    2016-10-01

    Small molecule-based regenerative engineering is emerging as a promising strategy for regenerating bone tissue. Small molecule cAMP analogues have been proposed as novel biofactors for bone repair and regeneration and, while promising, the effect that these small molecules have on angiogenesis, a critical requirement for successful bone regeneration, is still unclear. Our previous research demonstrated that the small molecule cAMP analogue 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) was able to promote initial osteoblast adhesion on a polymeric scaffold via cAMP signalling cascades. Here, we report that 8-Br-cAMP is capable of inducing in vitro cell-based VEGF production for angiogenesis promotion. We first demonstrated that treating osteoblast-like MC3T3-E1 cells with 8-Br-cAMP for 1 day significantly increased VEGF production and secretion. We then demonstrated that 8-Br-cAMP-induced cell-secreted VEGF is biologically active and may promote angiogenesis, as evidenced by increased human umbilical vein endothelial cells (HUVECs) migration and tubule formation. In addition, treatment of MC3T3-E1 cells with 8-Br-cAMP for as short as a single day resulted in enhanced ALP activity as well as matrix mineralization, demonstrating in vitro osteoblastic differentiation. A short-term 8-Br-cAMP treatment also addresses the concern of non-specific cytotoxicity, as our data indicate that a 1-day 8-Br-cAMP treatment scheme supports cellular proliferation of MC3T3-E1 cells as well as HUVECs. While the major concern associated with small molecule drugs is the risk of non-specific cytotoxicity, the short exposure treatment outlined in this paper provides a very promising strategy to mitigate the risk associated with small molecules. Copyright © 2013 John Wiley & Sons, Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-02

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

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

    Science.gov (United States)

    Guo, H H; Yu, C C; Sun, S X; Ma, X J; Yang, X C; Sun, K N; Jin, Q H

    2013-10-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Milovac, Dajana, E-mail: dmilovac@fkit.hr [Faculty of Chemical Engineering and Technology, University of Zagreb (Croatia); Gamboa-Martínez, Tatiana C. [Centre for Biomaterials and Tissue Engineering, Universitat Politècnica de València (Spain); Ivankovic, Marica [Faculty of Chemical Engineering and Technology, University of Zagreb (Croatia); Gallego Ferrer, Gloria [Centre for Biomaterials and Tissue Engineering, Universitat Politècnica de València (Spain); Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine, Valencia (Spain); Ivankovic, Hrvoje [Faculty of Chemical Engineering and Technology, University of Zagreb (Croatia)

    2014-09-01

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

  17. Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

    Science.gov (United States)

    Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells. PMID:28157196

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young-Hee; Bhattarai, Govinda [Department of Oral Biochemistry, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju (Korea, Republic of); Aryal, Santosh [Department of Bionanosystem Engineering, Chonbuk National University, Jeonju (Korea, Republic of); Lee, Nan-Hee [Department of Oral Biochemistry, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju (Korea, Republic of); Lee, Min-Ho [Department of Dental Biomaterials, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju (Korea, Republic of); Kim, Tae-Gun [Department of Conservative Dentistry, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Jhee, Eun-Chung [Department of Oral Biochemistry, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju (Korea, Republic of); Kim, Hak-Yong [Department of Bionanosystem Engineering, Chonbuk National University, Jeonju (Korea, Republic of); Yi, Ho-Keun, E-mail: yihokn@chonbuk.ac.kr [Department of Oral Biochemistry, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju (Korea, Republic of)

    2010-08-01

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

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

    Science.gov (United States)

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

    2010-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kihara, Tasuku [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Ichikawa, Saki [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yonezawa, Takayuki; Lee, Ji-Won [Research Institute for Biological Functions, Chubu University, Kasugai, Aichi (Japan); Akihisa, Toshihiro [College of Science and Technology, Nihon University, Tokyo (Japan); Woo, Je Tae [Research Institute for Biological Functions, Chubu University, Kasugai, Aichi (Japan); Michi, Yasuyuki; Amagasa, Teruo [Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan)

    2011-03-11

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

  2. Resveratrol inhibits the hydrogen dioxide-induced apoptosis via Sirt 1 activation in osteoblast cells.

    Science.gov (United States)

    He, Na; Zhu, Xuewei; He, Wei; Zhao, Shiwei; Zhao, Weiyan; Zhu, Chunlei

    2015-01-01

    Sirt 1 plays a critical role in stress responses. We determined the deregulation of Sirt 1 activity, p53 acetylation, Bcl-2 expression, and mitochondria-dependent apoptosis in mouse osteoblast MC3T3-E1 cells which were exposed to H2O2. And then we investigated the protective role of Sirt 1 activator, Resveratrol (RSV), against the H2O2-induced apoptosis. Results demonstrated that Sirt 1 and Bcl-2 were inhibited, whereas p53 acetylation, Bax, and caspase 9 were promoted by H2O2, as was aggravated by the Sirt 1 inhibitor, EX-527. Instead, RSV inhibited the H2O2-induced both p53 acetylation and the caspase 9 activation, whereas ameliorated the H2O2-induced Bcl-2 inhibition and apoptosis. In conclusion, Sirt 1 was downregulated during the H2O2-induced apoptosis in MC3T3-E1 cells. And the chemical activation of Sirt 1 inhibited the H2O2-induced apoptosis via the downregulation of p53 acetylation. Our results suggest that Sirt 1 upregulation appears to be an important strategy to inhibit the oxidative stress-induced apoptosis.

  3. Tumor necrosis factor-alpha influences cbfa1/runx2 gene expression in mouse osteoblasts%肿瘤坏死因子α干预小鼠成骨细胞cbfa1/runx2基因的表达

    Institute of Scientific and Technical Information of China (English)

    朱建华; 张沁; 刘继光

    2011-01-01

    BACKGROUND: Tumor necrosis factor-alpha (TNF-a) can decrease alkaline phosphatase (ALP) activity in periodontal ligamentfibroblasts and inhibit the functional transformation between periodontal ligament fibroblasts and osteoblasts.OBJECTIVE: To investigate the effects of TNF-a on the growth of mouse osteoblasts and cbfa1/runx2 gene expression.METHODS: Well growing mouse osteoblast line MC3T3/E1 were interfered with 20, 40, 60, 80 μg/L TNF-a. The normally culturedcells served as controls. cbfa1/runx2 mRNA expression in osteoblast line MC3T3/E1 was detected by RT-PCR. ALP activity wasdetermined by PNPP method and cell viability was measured by MTT assay.RESULTS AND CONCLUSION: cbfa1/runx2 mRNA expression was observed in the normally cultured osteoblast line MC3T3/E1.With increasing concentration of TNF-a concentration, cbfa1/runx2 mRNA expression, MC3T3/E1 cell viability and ALP activitywere gradually decreased. Results suggested that TNF-a can inhibit osteoblast line MC3T3/E1 growth and cbfa1/runx2 may beinvolved in osteoblast differentiation.%背景:肿瘤坏死因子α可降低牙周膜纤维细胞碱性磷酸酶的活性,抑制牙周膜纤维细胞向成骨细胞的功能转化.目的:观察肿瘤坏死因子α对小鼠成骨细胞生长及cbfa1/runx2基因表达的影响.方法:取生长良好的小鼠成骨细胞系MC3T3/E1细胞,分别以20,40,60,80 μg/L的肿瘤坏死因子α进行干预,以正常培养的细胞作为对照.采用RT-PCR法检测MC3T3/E1细胞cbfa1/runx2 mRNA的表达;PNPP法测定碱性磷酸酶活性;MTT法检测细胞活力.结果与结论:正常培养的MC3T3/E1细胞cbfa1/runx2 mRNA呈阳性表达,随着肿瘤坏死因子α浓度的增高,其表达水平逐渐下降.同时MC3T3/E1细胞活力和碱性磷酸酶活性也随肿瘤坏死因子α浓度的增高而下降.提示肿瘤坏死因子α可抑制MC3T3/E1细胞生长,而cbfa1/runx2可能参与了成骨细胞的分化过程.

  4. Effects of LPS-stimulated monocyte culture supernatant on the osteogenic function of osteoblastic cells in vitro%脂多糖刺激单核巨噬细胞培养上清液对成骨细胞成骨特性的影响

    Institute of Scientific and Technical Information of China (English)

    王海燕; 邓辉; 徐春燕; 叶洁; 胡荣党

    2011-01-01

    目的 探讨建立牙周炎症的体外实验模型的可行性,为后续进行牙周炎症状态下应力对牙槽骨改建的研究奠定基础.方法 以牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)脂多糖(lipopolysaccharide,LPS)Pg-LPS刺激小鼠单核巨噬细胞(RAW264.7)的培养上清液作用于体外培养的小鼠成骨细胞(MC3T3-E1)24 h和48 h后,分别通过噻唑蓝(MTT)法和碱性磷酸酶(ALP)试剂盒检测MC3T3-E1的增殖活性和碱性磷酸酶活性,观察脂多糖刺激的小鼠单核巨噬细胞培养上清液对小鼠成骨细胞成骨功能的影响.结果 Pg-LPS刺激RAW264.7后其上清液含L-1β、IL-6 和TNF-α等炎症因子,且与LPS的浓度和刺激时间呈依赖性.此上清液对MC3T3-E1的增殖和ALP活性均有抑制作用,亦与上清液中以上炎症因子的浓度呈依赖性,与体内牙周炎症状态相似.结论 Pg-LPS体外刺激RAW264.7的培养上清液可以应用于建立体外牙周炎症模型,这将为后续研究牙周炎症状态下应力对牙槽骨改建的影响奠定初步的基础.%Objective To investigate the influence of Pg-LPS stimulated monocyte (RAW264.7) culture supernatant on the proliferation and alkaline phosphatase (ALP) activity of osteoblastic cells (MC3T3-E1) in vitro. Methods The culture supernatant of monocytes stimulated with Pg-LPS was applied to osteoblasts MC3T3-E1 with different diluted concentrations(10%、20%、30%、40% and 50%) simultaneously for 24 h and 48 h in vitro.The cellular proliferation was assessed by MTT assay, Cellular ALP activity was examined using ALP measurement kit, and the results were statistically analyzed using SPSS 12.0 software package. Results LPS stimulated RAW264.7 to release inflammatory cytokines including IL-1β, IL-6 and TNF-α, whose secretion was in a time and dose dependent manner with the concentration of LPS. After the stimulation of different concentrations of inflammatory supernatant, the proliferation and ALP activity of MC3T3-El

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

    Science.gov (United States)

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

    2017-02-08

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

  6. Gene expression profiles and phosphorylation patterns of AMP-activated protein kinase subunits in various mesenchymal cell types

    Institute of Scientific and Technical Information of China (English)

    Wang Yugang; Fan Qiming; Ma Rui; Lin Wentao; Tang Tingting

    2014-01-01

    Background Recent studies on bone have shown an endocrine role of the skeleton,which could be impaired in various human diseases,including osteoporosis,obesity,and diabetes-associated bone diseases.As a sensor and regulator of energy metabolism,AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism.The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.Methods Reverse transcription-polymerase chain reaction (PCR) for relative quantification,real-time PCR for absolute quantification,and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types,including primary human mesenchymal stem cells (hMSCs) and hFOB,Saos-2,C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells.Results AMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types.AMPKY1 mRNA was abundantly expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 but not detected in human-derived cell types.AMPKY2 mRNA was mildly expressed in all cell types.AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells.AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2,in which AMPKβ2 protein overwhelmed AMPKβ1 expression.AMPKy1 and AMPKY2 proteins were expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells and only AMPKY2 protein was expressed in hMSCs,hFOB and Saos2 cells.AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.Conclusion The combination of AMPK α,β,and Y subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.

  7. Internalization of ferromagnetic nanowires by different living cells

    Directory of Open Access Journals (Sweden)

    Coey John

    2006-09-01

    Full Text Available Abstract The ability of living cells, either adherent or suspended, to internalize nickel nanowires is demonstrated for MC3T3-E1, UMR106-tumour and Marrow-Stromal cells. Nanowires were produced by electrodeposition, 20 μm long and 200 nm in diameter. Cell separation and manipulation was achieved for the three cell types. Applied magnetic field successfully oriented the internalized nanowires but no clear anisotropy is induced on the adherent cells. Nanowires tend to bind to cytoplasm metalloproteins and trigger lysosome reorganization around the nucleus. This work demonstrates the applications of nanowires in adherent and suspended cells for cell separation and manipulation, and further explore into their role in nanobiotechnology.

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

    Directory of Open Access Journals (Sweden)

    Lin H

    2014-07-01

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

  9. Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells.

    Science.gov (United States)

    Kim, Hak Jun; Shim, Hye Eun; Lee, Jun Hyuck; Kang, Yong-Cheol; Hur, Young Baek

    2015-12-28

    Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

  10. Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.

    Science.gov (United States)

    Cui, Yazhou; Luan, Jing; Li, Haiying; Zhou, Xiaoyan; Han, Jinxiang

    2016-01-01

    Mineralizing osteoblasts (MOBs) can release exosomes, although the functional significance remains unclear. In the present study, we demonstrate that exosomes derived from mineralizing pre-osteoblast MC3T3-E1 cells can promote bone marrow stromal cell (ST2) differentiation to osteoblasts. We reveal that MOB-derived exosomes significantly influence miRNA profiles in recipient ST2 cells, and these changes tend to activate the Wnt signaling pathway by inhibiting Axin1 expression and increasing β-catenin expression. We also suggest that MOB derived-exosomes partly induce the variation in miRNA expression in recipient ST2 cells by exosomal miRNA transfer. These findings suggest an exosome-mediated mode of cell-to-cell communication in the osteogenic microenvironment, and also indicate the potential of MOB exosomes in bone tissue engineering.

  11. Laser pulse dependent micro textured calcium phosphate coatings for improved wettability and cell compatibility.

    Science.gov (United States)

    Paital, Sameer R; He, Wei; Dahotre, Narendra B

    2010-07-01

    Surface wettability of an implant material is an important criterion in biological response as it controls the adsorption of proteins followed by attachment of cells to its surface. Hence, micro-textured calcium phosphate coatings with four length scales were synthesized on Ti-6Al-4V substrates by a laser cladding technique and their effects on wettability and cell adhesion were systematically evaluated. Microstructure and morphological evolutions of the coatings were studied using scanning electron and light optical microscopes respectively. The surface texture of coating defined in terms of a texture parameter was correlated to its wetting behavior. The contact angle of simulated body fluid measured by a static sessile drop technique, demonstrated an increased hydrophilicity with decreasing value of texture parameter. The influence of such textures on the in vitro bioactivity and in vitro biocompatibility were studied by the immersion of the samples in simulated body fluid and mouse MC3T3-E1 osteoblast-like cell culture respectively.

  12. Assessment of using laponite cross-linked poly(ethylene oxide) for controlled cell adhesion and mineralization.

    Science.gov (United States)

    Gaharwar, Akhilesh K; Schexnailder, Patrick J; Kline, Benjamin P; Schmidt, Gudrun

    2011-02-01

    The in vitro cytocompatibility of silicate (Laponite clay) cross-linked poly(ethylene oxide) (PEO) nanocomposite films using MC3T3-E1 mouse preosteoblast cells was investigated while cell adhesion, spreading, proliferation and mineralization were assessed as a function of film composition. By combining the advantageous characteristics of PEO polymer (hydrophilic, prevents protein and cell adhesion) with those of a synthetic and layered silicate (charged, degradable and potentially bioactive) some of the physical and chemical properties of the resulting polymer nanocomposites could be controlled. Hydration, dissolution and mechanical properties were examined and related to cell adhesion. Overall, this feasibility study demonstrates the ability of using model Laponite cross-linked PEO nanocomposites to create bioactive scaffolds.

  13. Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation

    Science.gov (United States)

    Zhi, J.; Sommerfeldt, D. W.; Rubin, C. T.; Hadjiargyrou, M.

    2001-01-01

    Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

  14. MicroRNA miR-378 regulates nephronectin expression modulating osteoblast differentiation by targeting GalNT-7.

    Directory of Open Access Journals (Sweden)

    Shireen Kahai

    Full Text Available MicroRNAs (miRNAs are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378* plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 3'-untranslated region (3'UTR contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 3'UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 3'UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7 for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation.

  15. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

  16. The influence of MicroRNA-150 in Osteoblast Matrix Mineralization.

    Science.gov (United States)

    Dong, Chun-Ling; Liu, Hao-Zhi; Zhang, Zhen-Chun; Zhao, Huan-Li; Zhao, Hui; Huang, Yan; Yao, Jian-Hua; Sun, Tian-Sheng

    2015-12-01

    This study investigated the influence of miR-150 expression on osteoblast matrix mineralization and its mechanisms. The mouse osteoblast cell line MC3T3-E1 was used as an in vitro model of bone formation. On the fifth day of mineralization, transfection experiments using agomiR-150, agomiR-NC, antagomiR-150 antagomiR-NC, and mock groups were set up to test the effects of miR-150 in MC3T3-E1 model. The mRNA and protein levels of OC, ALP, type I collagen, and OPN were measured by qRT-PCR and ELISA. Matrix mineralization was detected by alizarin red S (ARS) staining and flow cytometry was employed to quantify apoptosis in each group. RT-PCR and Western blot were applied to detect the expression of target gene MMP14. Our results demonstrated that the endogenous expression levels of miR-150, OC, ALP, type I collagen, and OPN in MC3T3-E1 cells increased steadily. Exogenous expressions of agomiR-150 and antagomiR-150 can significantly up-/down-regulate, respectively, the expression level of miR-150 in MC3T3-E1 cells. Compared with the mock group, higher expression levels of OC, ALP, type I collagen, and OPN mRNA were observed in the agomiR-150 group, while lower mRNA expression levels of OC, ALP, type I collagen, and OPN were found in the antagomiR-150 group. Based on these results, potential miR-150 targeted genes are discussed. Our results showed that miR-150 supports the osteoblastic phenotype related to osteoblast function and bone mineralization. Thus, miR-150 may have potential therapeutic applications in promoting bone formation in certain disease settings, such as in osteoporosis and in elderly patients.

  17. 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

  18. In vitro evaluation of poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether copolymer coating effects on cells adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rusen, Laurentiu [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania); Neacsu, Patricia; Cimpean, Anisoara [University of Bucharest, Department of Biochemistry and Molecular Biology, Bucharest (Romania); Valentin, Ion; Brajnicov, Simona; Dumitrescu, L.N. [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania); Banita, Janina [University of Bucharest, Faculty of Chemistry, Bucharest (Romania); IBAR, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, RO-060031 Bucharest (Romania); Dinca, Valentina, E-mail: valentina.dinca@inflpr.ro [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania); Dinescu, Maria [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania)

    2016-06-30

    Understanding and controlling natural and synthetic biointerfaces is known to be the key to a wide variety of application within cell culture and tissue engineering field. As both material characteristics and methods are important in tailoring biointerfaces characteristics, in this work we explore the feasibility of using Matrix Assisted Pulsed Laser Evaporation technique for obtaining synthetic copolymeric biocoatings (i.e. poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether) for evaluating in vitro Vero and MC3T3-E1 pre-osteoblasts cell response. Characterization and evaluation of the coated substrates were carried out using different techniques. The Fourier transform infrared spectroscopy data demonstrated that the main functional groups in the MAPLE-deposited films remained intact. Atomic Force Microscopy images showed the coatings to be continuous, with the surface roughness depending on the deposition parameters. Moreover, the behaviour of the coatings in medium mimicking the pH and temperature of the human body was studied and corelated to degradation. Spectro-ellipsometry (SE) and AFM measurements revealed the degradation trend during immersion time by the changes in coating thickness and roughness. In vitro biocompatibility was studied by indirect contact tests on Vero cells in accordance with ISO 10993-5/2009. The results obtained in terms of cell morphology (phase contrast microscopy) and cytotoxicity (LDH and MTT assays) proved biocompatibility. Furthermore, direct contact assays on MC3T3-E1 pre-osteoblasts demonstrated the capacity of all analyzed specimens to support cell adhesion, normal cellular morphology and growth.

  19. In vitro evaluation of poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether copolymer coating effects on cells adhesion and proliferation

    Science.gov (United States)

    Rusen, Laurentiu; Neacsu, Patricia; Cimpean, Anisoara; Valentin, Ion; Brajnicov, Simona; Dumitrescu, L. N.; Banita, Janina; Dinca, Valentina; Dinescu, Maria

    2016-06-01

    Understanding and controlling natural and synthetic biointerfaces is known to be the key to a wide variety of application within cell culture and tissue engineering field. As both material characteristics and methods are important in tailoring biointerfaces characteristics, in this work we explore the feasibility of using Matrix Assisted Pulsed Laser Evaporation technique for obtaining synthetic copolymeric biocoatings (i.e. poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether) for evaluating in vitro Vero and MC3T3-E1 pre-osteoblasts cell response. Characterization and evaluation of the coated substrates were carried out using different techniques. The Fourier transform infrared spectroscopy data demonstrated that the main functional groups in the MAPLE-deposited films remained intact. Atomic Force Microscopy images showed the coatings to be continuous, with the surface roughness depending on the deposition parameters. Moreover, the behaviour of the coatings in medium mimicking the pH and temperature of the human body was studied and corelated to degradation. Spectro-ellipsometry (SE) and AFM measurements revealed the degradation trend during immersion time by the changes in coating thickness and roughness. In vitro biocompatibility was studied by indirect contact tests on Vero cells in accordance with ISO 10993-5/2009. The results obtained in terms of cell morphology (phase contrast microscopy) and cytotoxicity (LDH and MTT assays) proved biocompatibility. Furthermore, direct contact assays on MC3T3-E1 pre-osteoblasts demonstrated the capacity of all analyzed specimens to support cell adhesion, normal cellular morphology and growth.

  20. Knowledge discovery of cell-cell and cell-surface interactions

    Science.gov (United States)

    Su, Jing

    High-throughput cell culture is an emerging technology that shows promise as a tool for research in tissue engineering, drug discovery, and medical diagnostics. An important, but overlooked, challenge is the integration of experimental methods with information processing suitable for handling large databases of cell-cell and cell-substrate interactions. In this work the traditional global descriptions of cell behaviors and surface characteristics was shown insufficient for investigating short-distance cell-to-cell and cell-to-surface interactions. Traditional summary metrics cannot distinguish information of cell near neighborhood from the average, global features, thus often is not suitable for studying distance-sensitive cell behaviors. The problem of traditional summary metrics was addressed by introducing individual-cell based local metrics that emphasize cell local environment. An individual-cell based local data analysis method was established. Contact inhibition of cell proliferation was used as a benchmark for the effectiveness of the local metrics and the method. Where global, summary metrics were unsuccessful, the local metrics successfully and quantitatively distinguished the contact inhibition effects of MC3T3-E1 cells on PLGA, PCL, and TCPS surfaces. In order to test the new metrics and analysis method in detail, a model of cell contact inhibition was proposed. Monte Carlo simulation was performed for validating the individual-cell based local data analysis method as well as the cell model itself. The simulation results well matched with the experimental observations. The parameters used in the cell model provided new descriptions of both cell behaviors and surface characteristics. Based on the viewpoint of individual cells, the local metrics and local data analysis method were extended to the investigation of cell-surface interactions, and a new high-throughput screening and knowledge discovery method on combinatorial libraries, local cell

  1. Storage of cell lines.

    Science.gov (United States)

    Parker, Katharine A

    2011-01-01

    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

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

    Directory of Open Access Journals (Sweden)

    Chen Chien-Chiang

    2009-10-01

    Full Text Available Abstract Background The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs, which addresses this shortcoming. Results The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. Conclusion LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical

  3. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  4. Synthesis and in vitro bioactivity of mesoporous bioactive glass scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Shih, C.J., E-mail: cjshih@kmu.edu.tw [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chen, H.T. [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Huang, L.F. [School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lu, P.S.; Chang, H.F. [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, I.L., E-mail: 84004@cch.org.tw [Department of Orthopaedic Surgery, Chang-Hua Christian Hospital, Changhua 500, Taiwan (China)

    2010-06-15

    The main objective of the present study was to determine the effect of thermal treatment procedures (calcination temperature, heating rate and duration time) on the synthesis of SiO{sub 2}-CaO-P{sub 2}O{sub 5} mesoporous bioactive glass scaffolds. This is accomplished by thermogravimetric analyses, Fourier transform infrared (FTIR) absorption spectra, X-ray diffraction (XRD) and by analysis of nitrogen adsorption/desorption isotherms. In vitro bioactivity can also be assessed by the cytotoxic effect of the glasses on the NIH-3T3 cell line, and by characterization of MC-3T3-E1 cell attachment.

  5. Dynamic cell adhesion and migration on nanoscale grooved substrates.

    Science.gov (United States)

    Lamers, E; te Riet, J; Domanski, M; Luttge, R; Figdor, C G; Gardeniers, J G E; Walboomers, X F; Jansen, J A

    2012-01-01

    Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

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

    Science.gov (United States)

    Pina, S; Vieira, S I; Rego, P; Torres, P M C; da Cruz e Silva, O A B; da Cruz e Silva, E F; Ferreira, J M F

    2010-09-07

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

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

    Directory of Open Access Journals (Sweden)

    S Pina

    2010-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Giovanni Tulipano

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    JunFeng Dong

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-01

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

  11. Sirt1 overexpression protects murine osteoblasts against TNF-α-induced injury in vitro by suppressing the NF-KB signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Wei HUANG; Wei-lin SHANG; Hua-dong WANG; Wen-wen WU; Shu-xun HOU

    2012-01-01

    Sirtuin 1 (Sirt1) is the class III histone/protein deacetylase that interferes with the NF-KB signaling pathway,thereby has antiinflammatory function.This study was undertaken to investigate whether Sirt1 could protect osteoblasts against TNF-α-induced injury in vitro.Methods:Murine osteoblastic cell line,MC3T3-E1,was used.Overexpress of Sirt1 protein in MC3T3-E1 cells was made by transfection the cells with Sirt1-overexpressing adenovirus.The levels of mRNAs and proteins were determined with qRT-PCR and Western blotting,respectively.The activity of NF-κB was examined using NF-κB luciferase assay.The NO concentration was measured using the Griess method.Results:Treatment of MC3T3-E1 cells with TNF-α (2.5-10 ng/mL) suppressed Sirt1 protein expression in a concentration-dependent manner.TNF-α (5 ng/mL) resulted in an increase in apoptosis and a reduction in ALP activity in the cells.Overexpression of Sirt1in the cells significantly attenuated TNF-α-induced injury through suppressing apoptosis,increasing ALP activity,and increasing the expression of Runx2 and osteocalcin mRNAs.Furthermore,overexpression of Sirt1 in the cells significantly suppressed TNF-α-induced NF-κB activation,followed by reducing the expression of iNOS and NO formation.Sirt1 activator resveratrol (10 μmol/L) mimicked the protection of the ceils by Sirt1 overexpression against TNF-α-induced injury,which was reversed by the Sirt1 inhibitor EX-527 (5μmol/L).Conclusion:Overexpression of Sirt1 protects MC3T3-E1 osteoblasts aganst TNF-α-induced cell injury in vitro,at least in part,via suppressing NF-KB signaling.Sirt1 may be a novel therapeutic target for treating rheumatoid arthritis-related bone loss.

  12. 聚乳酸-羟基乙酸共聚物-多壁碳纳米管复合膜诱导成骨细胞分化的机制%Mechanism underlying differentiation of osteoblast on poly-(lactic-co-glycolic acid) and carboxylated multiwall nanocomposite films

    Institute of Scientific and Technical Information of China (English)

    聂力; 张光明

    2015-01-01

    Objective To investigate the mechanism underlying the response of osteoblast cell line MC3T3-E1 cultured on poly-(lactic-co-glycolic acid) and carboxylated multiwall (PLGA-MWCNTs) nanocomposite films.Methods After the MC3T3-E1 cells were cultured on PLGA-MWCNTs,poly-(lacticco-glycolic acid) film (PLGA) and tissue culture polystyrene (TCPS) for 72 h,RT-PCR was performed to determine the expression of bone-specific genes,including the alkaline phosphatase (ALP),bone sialoprotein (BSP) and osteocalcin (OCN).Western blotting,and immunofluorescence analysis was used to examine the cell viability and the expression of focal adhesion kinase (FAK).The expression of p-ERK1/2,pJNK and p-p38 MAPK proteins in each group of MC3T3-E1 cells was determined by Western blotting.Then,the three groups of MC3T3-E1 cells were added with 30 mmol/L PD98059 (a specific ERK1/2 inhibitor).After culture for 72 h,stained with MTT,Live/Dead and Texas Red-labeled phalloidin,the cells were observed for cell viability and production of actin fiber under confocal laser scanning microscope.Results The mRNA levels of ALP,BSP,and OCN in MC3T3-E1 cells on PLGA-MWCNTs films were higher compared with those on PLGA or TCPS (all P<0.05),whereas these levels did not differ significantly between cells on PLGA and on TCPS (P>0.05).The staining pattern of pFAK for MC3T3-T1 cells cultured on PLGA-MWCNTs films were more evenly distributed when compared with those on PLGA or TCPS,and the expression level of pFAK in cells on PLGA-MWCNTs films (2.3±0.3) was much higher than that in cells on PLGA (1.4±0.2) or TCPS (1.0±0.2) (all P<0.05).The expression level of p-ERK 1/2 in MC3T3-E1 cells on PLGA-MWCNTs films (3.3±0.2) were nearly two times higher than that in the cells on PLGA (1.3±0.2) or TCPS (1.0±0.2) (all P<0.05).There was no statistical difference in expression of p-JNK or p-p38 MAPK among groups (all P>0.05).After culture with the ERK1/2 inhibitor PD98059 for 72 h,the cell viability and actin

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Cell-laden poly(ɛ-caprolactone)/alginate hybrid scaffolds fabricated by an aerosol cross-linking process for obtaining homogeneous cell distribution: fabrication, seeding efficiency, and cell proliferation and distribution.

    Science.gov (United States)

    Lee, HyeongJin; Ahn, SeungHyun; Bonassar, Lawrence J; Chun, Wook; Kim, GeunHyung

    2013-10-01

    Generally, solid-freeform fabricated scaffolds show a controllable pore structure (pore size, porosity, pore connectivity, and permeability) and mechanical properties by using computer-aided techniques. Although the scaffolds can provide repeated and appropriate pore structures for tissue regeneration, they have a low biological activity, such as low cell-seeding efficiency and nonuniform cell density in the scaffold interior after a long culture period, due to a large pore size and completely open pores. Here we fabricated three different poly(ɛ-caprolactone) (PCL)/alginate scaffolds: (1) a rapid prototyped porous PCL scaffold coated with an alginate, (2) the same PCL scaffold coated with a mixture of alginate and cells, and (3) a multidispensed hybrid PCL/alginate scaffold embedded with cell-laden alginate struts. The three scaffolds had similar micropore structures (pore size = 430-580 μm, porosity = 62%-68%, square pore shape). Preosteoblast cells (MC3T3-E1) were used at the same cell density in each scaffold. By measuring cell-seeding efficiency, cell viability, and cell distribution after various periods of culturing, we sought to determine which scaffold was more appropriate for homogeneously regenerated tissues.

  15. An efficient surface modification using 2-methacryloyloxyethyl phosphorylcholine to control cell attachment via photochemical reaction in a microchannel.

    Science.gov (United States)

    Jang, Kihoon; Sato, Kae; Tanaka, Yo; Xu, Yan; Sato, Moritoshi; Nakajima, Takahiro; Mawatari, Kazuma; Konno, Tomohiro; Ishihara, Kazuhiko; Kitamori, Takehiko

    2010-08-07

    This report describes a direct approach for cell micropatterning in a closed glass microchannel. To control the cell adhesiveness inside the microchannel, the application of an external stimulus such as ultraviolet (UV) was indispensible. This technique focused on the use of a modified 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to be a non-biofouling compound that is a photocleavable linker (PL), to localize cells via connection to an amino-terminated silanized surface. Using UV light illumination, the MPC polymer was selectively eliminated by photochemical reaction that controlled the cell attachment inside the microchannel. For suitable cell micropatterning in a microchannel, the optimal UV illumination time and concentration for cell suspension were investigated. After selective removal of the MPC polymer through the photomask, MC-3T3 E1 cells and vascular endothelial cells (ECs) were localized only to the UV-exposed area. In addition, the stability of patterned ECs was also confirmed by culturing for 2 weeks in a microchannel under flow conditions. Furthermore, we employed two different types of cells inside the same microchannel through multiple removal of the MPC polymer. ECs and Piccells were localized in both the upper and down streams of the microchannel, respectively. When the ECs were stimulated by adenosine triphosphate (ATP), NO was secreted from the ECs and could be detected by fluorescence resonance energy transfer (FRET) in Piccells, which is a cell-based NO indicator. This technique can be a powerful tool for analyzing cell interaction research.

  16. Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

    Science.gov (United States)

    Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

    2014-09-01

    The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications.

  17. Improvement of cell adhesion on poly(L-lactide) by atmospheric plasma treatment.

    Science.gov (United States)

    Nakagawa, Masafumi; Teraoka, Fumio; Fujimoto, Shinji; Hamada, Yoshinosuke; Kibayashi, Hiroyuki; Takahashi, Junzo

    2006-04-01

    The purpose of this study is to elucidate the interaction between the cell and the surface of poly(L-lactide) (PLLA) samples, which were modified using a low-temperature plasma treatment apparatus at atmospheric pressure. The plasma treatments were carried out in the atmospheres of air, carbon dioxide (CO2), and perfluoro propane (C3F8) gas. The PLLA samples before and after the plasma treatment were analyzed by XPS and their contact angles with water. Furthermore, the cell adhesion capability and cell mass culturing tests on the PLLA samples were carried out using MC3T3-E1 cells. The results showed that the contact angle of the samples, which was plasma treated in air or in CO2 gas, decreased compared with that of the untreated samples. On the other hand, the contact angle of the samples, which was plasma treated in the C3F8 gas, increased compared with the untreated plasma samples. The cell response on the PLLA samples plasma treated in air or in the CO2 gas were significantly superior to that of the PLLA samples, which was plasma treated in the C3F8 gas.

  18. Structure design and fabrication of porous hydroxyapatite microspheres for cell delivery

    Science.gov (United States)

    Li, Ruijing; Chen, Kexin; Li, Geng; Han, Guoxiang; Yu, Sheng; Yao, Juming; Cai, Yurong

    2016-09-01

    Porous microspheres fabricated from bioceramics have great potential for cell delivery in injectable tissue engineering application. The size and structure of pores in the microspheres are important for the effective protection and transportation of cells. In this study, porous hydroxyapatite microspheres are fabricated through the water-in-oil emulsion method followed by a calcination treatment at the high temperature. Both self-made resorcinol-formaldehyde (RF) composite spheres and camphene are used as pore-forming agents to produce big pores corresponding to the size of RF spheres and connected channel among big pores in hydroxyapatite matrix. The properties of the microspheres are characterized using X-ray diffraction, thermogravimetry analysis, universal material machine, field emission scanning electron microscopy. Cell assays are carried out to evaluate the cellular compatibility of the microspheres. The results showed that the hydroxyapatite microspheres with controllable pore structure and high porosity could be fabricated by this method, which have better strength to resist the compressive force. The microspheres are conducive to support adhesion, proliferation and differentiation of MC3T3-E1 cells. The results indicate that the obtained porous hydroxyapatite microspheres can be a permeable microenvironment for cell delivery in injectable tissue engineering.

  19. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

  20. Eu/Tb codoped spindle-shaped fluorinated hydroxyapatite nanoparticles for dual-color cell imaging.

    Science.gov (United States)

    Ma, Baojin; Zhang, Shan; Qiu, Jichuan; Li, Jianhua; Sang, Yuanhua; Xia, Haibing; Jiang, Huaidong; Claverie, Jerome; Liu, Hong

    2016-06-02

    Lanthanide doped fluorinated hydroxyapatite (FAp) nanoparticles are promising cell imaging nanomaterials but they are excited at wavelengths which do not match the light sources usually found in a commercial confocal laser scanning microscope (CLSM). In this work, we have successfully prepared spindle-shaped Eu/Tb codoped FAp nanoparticles by a hydrothermal method. Compared with single Eu doped FAp, Eu/Tb codoped FAp can be excited by a 488 nm laser, and exhibit both green and red light emission. By changing the amounts of Eu and Tb peaks, the emission in the green region (500-580 nm) can be decreased to the benefit of the emission in the red region (580-720 nm), thus reaching a balanced dual color emission. Using MC3T3-E1 cells co-cultured with Eu/Tb codoped FAp nanoparticles, it is observed that the nanoparticles are cytocompatible even at a concentration as high as 800 μg ml(-1). The Eu/Tb codoped FAp nanoparticles are located in the cytoplasm and can be monitored by dual color-green and red imaging with a single excitation light at 488 nm. At a concentration of 200 μg ml(-1), the cytoplasm is saturated in 8 hours, and Eu/Tb codoped FAp nanoparticles retain their fluorescence for at least 3 days. The cytocompatible Eu/Tb codoped FAp nanoparticles with unique dual color emission will be of great use for cell and tissue imaging.

  1. Vitamin D(3) enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts.

    Science.gov (United States)

    Tsuji, K; Kraut, N; Groudine, M; Noda, M

    2001-05-28

    I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D(3), specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D(3) upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D(3) increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D(3) enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D(3).

  2. Green Synthesis of Red-Emitting Carbon Nanodots as a Novel "Turn-on" Nanothermometer in Living Cells.

    Science.gov (United States)

    Wang, Chuanxi; Jiang, Kaili; Wu, Qian; Wu, Jiapeng; Zhang, Chi

    2016-10-01

    Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel "turn-on" carbon-dot-based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave-assisted method and exhibit red fluorescence (λem =615 nm) with high quantum yields (15 %). Then, an on-off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation-induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs-GSH mixture could behave as an off-on fluorescent probe for temperature. Thus, red-emitting CNDs can be utilized for "turn-on" fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3-E1 cells as an example model to demonstrate the red-emitting CNDs can function as "non-contact" tools for the accurate measurement of temperature and its gradient inside a living cell.

  3. Suppression of Wnt signaling by Dkk1 attenuates PTH-mediated stromal cell response and new bone formation.

    Science.gov (United States)

    Guo, Jun; Liu, Minlin; Yang, Dehong; Bouxsein, Mary L; Saito, Hiroaki; Galvin, R J Sells; Kuhstoss, Stuart A; Thomas, Clare C; Schipani, Ernestina; Baron, Roland; Bringhurst, F Richard; Kronenberg, Henry M

    2010-02-03

    Parathyroid hormone (PTH) suppresses Dickkopf 1 (Dkk1) expression in osteoblasts. To determine whether this suppression is essential for PTH-mediated Wnt signaling and bone formation, we examined mice that overexpress Dkk1 in osteoblasts (Dkk1 mice). Dkk1 mice were osteopenic due to abnormal osteoblast and osteoclast activity. When fed a low-calcium diet, and in two other models of hyperparathyroidism, these mice failed to develop the peritrabecular stromal cell response ("osteitis fibrosis") and new bone formation seen in wild-type mice. Despite these effects of Dkk1 overexpression, PTH still activated Wnt signaling in Dkk1 mice and in osteoblastic cells cultured from these mice. In cultured MC3T3E1 preosteoblastic cells, PTH dramatically suppressed Dkk1 expression, induced PKA-mediated phosphorylation of beta-catenin, and significantly enhanced Lef1 expression. Our findings indicate that the full actions of PTH require intact Wnt signaling but that PTH can activate the Wnt pathway despite overexpression of Dkk1.

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

    Science.gov (United States)

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

    2014-11-01

    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 were improved effectively on GRGD-modified PEEK surface. PEEK modified with GRGD on its surface has potential use in orthopedic or dental implants.

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

    Directory of Open Access Journals (Sweden)

    Yeong-Min Yoo

    2014-04-01

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

  6. Generation of microgrooved silica nanotube membranes with sustained drug delivery and cell contact guidance ability by using a Teflon microfluidic chip

    Directory of Open Access Journals (Sweden)

    Song Chen, Xuetao Shi, Shanmugavel Chinnathambi, Hongkai Wu and Nobutaka Hanagata

    2013-01-01

    Full Text Available Silica nanotubes have been extensively applied in the biomedical field. However, very little attention has been paid to the fabrication and application of micropatterned silica nanotubes. In the present study, microgrooved silica nanotube membranes were fabricated in situ by microgrooving silica-coated collagen hybrid fibril hydrogels in a Teflon microfluidic chip followed by calcination for removal of collagen fibrils. Scanning electron microscopy images showed that the resulting silica nanotube membranes displayed a typical microgroove/ridge surface topography with ~50 μm microgroove width and ~120 μm ridge width. They supported adsorption of bone morphogenetic protein 2 (BMP-2 and exhibited a sustained release behavior for BMP-2. After culturing with osteoblast MC3T3-E1 cells, they induced an enhanced osteoblast differentiation due to the release of biologically active BMP-2 and a strong contact guidance ability to directly align and elongate osteoblasts due to the presence of microgrooved surface topography, indicating their potential application as a multi-functional cell-supporting matrix for tissue generation.

  7. Effects of total flavonoids from Drynaria fortunei on the proliferation and osteogenic differentiation of rat dental pulp stem cells.

    Science.gov (United States)

    Huang, Xiao-Fei; Yuan, Su-Jian; Yang, Cheng

    2012-09-01

    Dental pulp stem cells (DPSCs) have the potential to form bone, nerve and fat, and are a candidate for use in regenerative medicine. Previous studies indicated that total flavonoids from Drynaria fortunei show a stimulative effect on the proliferation and osteogenic differentiation of osteoblastic MC3T3-E1 cells in vitro. This study aimed to investigate the effect of total flavonoids from Drynaria fortunei on the proliferation and osteogenic differentiation of rat DPSCs, and to further clarify the mechanisms involved. DPSCs were isolated by enzymatic digestion and identified using the CD44, CD29 and CD34 markers by immunohistochemistry, and exposed to 0.01, 0.05 and 0.1 g/l total flavonoids from Drynaria fortunei media. Total flavonoids from Drynaria fortunei promoted the proliferation of DPSCs in a dose-dependent manner and this effect may depend on the shortening of the G0/G1 phase and promotion of the S phase. Compared with the control group, the levels of alkaline phosphatase (ALP) and the expression of osteogenic genes increased with the concentrations of total flavonoids from Drynaria fortunei, and the volume and number of calcified nodules in the Drynaria groups was bigger compared to the control group. These results suggest that total flavonoid from Drynaria fortunei directly stimulates DPSC proliferation and osteogenic differentiation, and may serve as a new promising candidate drug for dental tissue engineering and bone regeneration.

  8. Generation of microgrooved silica nanotube membranes with sustained drug delivery and cell contact guidance ability by using a Teflon microfluidic chip

    Science.gov (United States)

    Chen, Song; Shi, Xuetao; Chinnathambi, Shanmugavel; Wu, Hongkai; Hanagata, Nobutaka

    2013-02-01

    Silica nanotubes have been extensively applied in the biomedical field. However, very little attention has been paid to the fabrication and application of micropatterned silica nanotubes. In the present study, microgrooved silica nanotube membranes were fabricated in situ by microgrooving silica-coated collagen hybrid fibril hydrogels in a Teflon microfluidic chip followed by calcination for removal of collagen fibrils. Scanning electron microscopy images showed that the resulting silica nanotube membranes displayed a typical microgroove/ridge surface topography with ˜50 μm microgroove width and ˜120 μm ridge width. They supported adsorption of bone morphogenetic protein 2 (BMP-2) and exhibited a sustained release behavior for BMP-2. After culturing with osteoblast MC3T3-E1 cells, they induced an enhanced osteoblast differentiation due to the release of biologically active BMP-2 and a strong contact guidance ability to directly align and elongate osteoblasts due to the presence of microgrooved surface topography, indicating their potential application as a multi-functional cell-supporting matrix for tissue generation.

  9. The effects of substrate-streching strain on the BMP-2 mRNA expression in three kinds of mouse cell lines%基底拉伸应变对小鼠三种骨组织细胞BMP-2 mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    闫玉仙; 宋梅; 郭春; 郭勇; 宫元伟; 李瑞欣; 张西正

    2010-01-01

    目的 研究基底拉伸应变对小鼠成骨细胞系MC3T3-E1、破骨细胞系RAW264.7及骨细胞MLO-Y4三种细胞BMP-2 mRNA表达的影响.方法 三种细胞随机分为0 με、1 000 με、1 500 με、2 000 με、2 500 με和5 000 με组,最佳拉伸时间和周期为1次/d,每次1 h,连续3 d,频率为0.5 Hz.采用卫生装备研究所自行设计研制的四点弯曲装置对小鼠三种细胞进行拉伸加载.采用RT-PCR技术分别研究不同应变对小鼠三种细胞BMP-2 mRNA表达.结果 MC3T3-E1细胞RT-PCR结果显示:1 500 με、2 000 με组和2 500 με组与0 με组相比BMP-2 mRNA表达显著增强(P<0.01);5 000 με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);RAW264.7细胞RT-PCR结果显示:1 500 με、2 000 με组和2 500 με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);5 000με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);MLO-Y4细胞BMP2基因表达结果与MC3T3-E1一致.结论 ①BMP-2在成骨细胞系MC3T3-E1、破骨细胞系RAW-264.7及骨细胞系MLO-Y4三种细胞中均有表达;②1 500 με、2 000 με、2 500 με三种生理剂量的拉伸应变可以显著增加MC3T3-E1、MLO-Y4 细胞BMP-2的表达,并呈剂量依赖性,超生理剂量5 000 με可以显著降低MC3T3-E1、MLO-Y4细胞BMP-2的表达;③相同的力学拉伸作用条件下,BMP-2在RAW-264.7细胞中表达与MC3T3-E1、MLO-Y4细胞的表达趋势相反.

  10. Molluscan cells in culture: primary cell cultures and cell lines.

    Science.gov (United States)

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  11. Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

    Science.gov (United States)

    Kitami, Megumi; Kaku, Masaru; Rocabado, Juan Marcelo Rosales; Ida, Takako; Akiba, Nami; Uoshima, Katsumi

    2016-09-01

    Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc.

  12. Dynamic cell adhesion and migration on nanoscale grooved substrates

    Directory of Open Access Journals (Sweden)

    E Lamers

    2012-03-01

    Full Text Available Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

  13. Effect of sonicated extracts of Porphyromonas gingivalis on osteogenic differentiation of mouse osteoblasts%牙龈卟啉单胞菌超声提取物对小鼠成骨细胞骨向分化蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    张剑英; 俞少杰; 付云

    2013-01-01

    Objective To investigate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on osteogenic differentiation of mouse osteoblast cell line MC3T3-E1.Methods PgW83 was cultured under standard anaerobic conditions and extracted by sonication.Mouse osteoblast cell line MC3T3-E1 was cultured with various concentrations of the extraction(0,10,100,1000 mg/L).Western blotting was applied to investigate the expression of osteocalcin(OC),bone sialoprotein(BSP),osteopo ntin(OPN)and osteonectin(ON).The activity of alkaline phosphatase (ALP) was detected by microplate reader after 14 days.Mineralization nodule formation was measured by alizarin red staining after 21 days.Results Compared with the control group,the extracts of Pg decreased OC and ON expression in a dose-dependent manner(OC relative expression:1.000 ± 0.000,0.852 ± 0.110,0.625 ± 0.451,0.213 ± 0.053),(ON relative expression:1.000 ± 0.000,1.035 ± 0.133,0.141 ± 0.023,0.020 ± 0.003) (P < 0.05).The expression of OPN was down-regulated significantly in MC3T3-E1 treated with 1000 mg/L extraction (0.572 ± 0.162) compared with control group,10 and 100 mg/L (1.000 ± 0.000,1.029 ± 0.135,1.199 ± 0.337) (P < 0.05).The expression of BSP remained unchanged when the cells were cultured with or without extraction (BSP relative expression:1.000 ± 0.000,0.831 ± 0.182,0.897 ± 0.115,0.778 ± 0.235) (P >0.05).Meanwhile,the extracts of Pg decreased ALP activity [control group:(0.0275 ±0.0014) U/gprot,10 mg/L:(0.0140 ±0.0011) U/gprot,100 mg/L:(0.0057 ±0.0013) U/gprot,1000 mg/L:(0.0020 ± 0.0008) U/gprot] (P < 0.05) and reduced mineralization nodule formation.Conclusions The results suggest that Pg may inhibit osteoblasts'osteogenic function by down-regulation of osteogenic differentiation related proteins.%目的 研究牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)W83超声提取物对小鼠颅顶前骨细胞亚克隆14(MC3T3-E1)骨向分化相关蛋白表达的影响,探讨Pg超声提取物

  14. 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.

  15. An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds

    Directory of Open Access Journals (Sweden)

    Alexander Röder

    2015-11-01

    Full Text Available The use of biopolymers as a three dimensional (3D support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface properties to assess whether seeding efficiencies and cell growth on 3D scaffolds were affected. Cell attachment and growth of murine calvarial osteoblast (MC3T3-E1 cells within a melt-electrospun poly-ε-caprolactone scaffold were assessed when cultured in either “low-adhesive” non-treated or corona discharged-treated well-plates. Increased cell adhesion was observed on the scaffold placed in the surface treated culture plates compared to the scaffold in the non-treated plates 24 h after seeding, although it was not significant. However, higher cell metabolic activity was observed on the bases of all well-plates than on the scaffold, except for day 21, well metabolic activity was higher in the scaffold contained in non-treated plate than the base. These results indicate that there is no advantage in using non-treated plates to improve initial cell seeding in 3D polymeric tissue engineering scaffolds, however non-treated plates may provide an improved metabolic environment for long-term studies.

  16. Modification of gellan gum with nanocrystalline hydroxyapatite facilitates cell expansion and spontaneous osteogenesis.

    Science.gov (United States)

    Jamshidi, Parastoo; Chouhan, Gurpreet; Williams, Richard L; Cox, Sophie C; Grover, Liam M

    2016-07-01

    Nanocomposites composed of hydrogels and calcium phosphates are of great interest in the development of bone graft replacements since they may have a structural and compositional resemblance to bone. Culture beads formed from such materials could be used in stirred tank culture and thereby enable cell expansion in a sufficiently efficient manner to allow for the generation of enough large number of cells for large-scale bone reconstruction. Although combinations of materials such as alginate, collagens, and various calcium phosphates have been investigated as culture beads, these materials are unsuitable for application since they have been shown to rapidly degrade in physiological conditions and enable relatively little tailoring of mechanical properties. In this study, gellan gum-nano sized hydroxyapatite (nHA) composites, which have been shown to be resistant to degradation and easily modified with respect to modulus, were formulated and characterized as regards their ability to enable cell attachment and proliferation. It was shown that the addition of 5 wt% of nHA to the culture beads enabled cell attachment and that an increase in nHA concentration to up to 25 wt% enhanced the rate of cell proliferation. Most importantly, it was demonstrated that the addition of nHA to the cell culture beads enabled the formation of nodules in culture of MC3T3-E1 cells and strikingly stimulated the osteogenic differentiation of bone marrow stromal cells in the absence of osteogenic media when compared with tissue culture plastic (TCP) with the same condition. Biotechnol. Bioeng. 2016;113: 1568-1576. © 2016 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    K Gellynck

    2013-06-01

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

  18. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

  19. SiO2/TiO2 Nanocomposite Films on Polystyrene for Light-Induced Cell Detachment Application.

    Science.gov (United States)

    Cheng, Zhiguo; Cheng, Kui; Weng, Wenjian

    2017-01-25

    Light-induced cell detachment shows much potential in in vitro cell culture and calls for high-performance light-responsive films. In this study, a smooth and dense SiO2/TiO2 nanocomposite thin film with thickness of around 250 nm was first fabricated on H2O2 treated polystyrene (PS) substrate via a low-temperature sol-gel method. It was observed that the film could well-adhere on the PS surface and the bonding strength became increasingly high with the increase of SiO2 content. The peeling strength and shear strength reached 3.05 and 30.02 MPa, respectively. It was observed the surface of the film could transform into superhydrophilic upon 20 min illumination of ultraviolet with a wavelength of 365 nm (UV365). In cell culture, cells, i.e., NIH3T3 and MC3T3-E1 cells, cultured on SiO2/TiO2 nanocomposite film were easily detached after 10 min of UV365 illumination; the detachment rates reached 90.8% and 88.6%, respectively. Correspondingly, continuous cell sheets with good viability were also easily obtained through the same way. The present work shows that SiO2/TiO2 nanocomposite thin film could be easily prepared on polymeric surface at low temperature. The corresponding film exhibits excellent biocompatibility, high bonding strength, and good light responses. It could be a good candidate for the surface of cell culture utensils with light-induced cell detachment property.

  20. Development of multi-walled carbon nanotubes reinforced monetite bionanocomposite cements for orthopedic applications.

    Science.gov (United States)

    Boroujeni, Nariman Mansoori; Zhou, Huan; Luchini, Timothy J F; Bhaduri, Sarit B

    2013-10-01

    In this study, we present results of our research on biodegradable monetite (DCPA, CaHPO4) cement with surface-modified multi-walled carbon nanotubes (mMWCNTs) as potential bone defect repair material. The cement pastes showed desirable handling properties and possessed a suitable setting time for use in surgical setting. The incorporation of mMWCNTs shortened the setting time of DCPA and increased the compressive strength of DCPA cement from 11.09±1.85 MPa to 21.56±2.47 MPa. The cytocompatibility of the materials was investigated in vitro using the preosteoblast cell line MC3T3-E1. An increase of cell numbers was observed on both DCPA and DCPA-mMWCNTs. Scanning electron microscopy (SEM) results also revealed an obvious cell growth on the surface of the cements. Based on these results, DCPA-mMWCNTs composite cements can be considered as potential bone defect repair materials.

  1. Additive manufacturing of wet-spun polymeric scaffolds for bone tissue engineering.

    Science.gov (United States)

    Puppi, Dario; Mota, Carlos; Gazzarri, Matteo; Dinucci, Dinuccio; Gloria, Antonio; Myrzabekova, Mairam; Ambrosio, Luigi; Chiellini, Federica

    2012-12-01

    An Additive Manufacturing technique for the fabrication of three-dimensional polymeric scaffolds, based on wet-spinning of poly(ε-caprolactone) (PCL) or PCL/hydroxyapatite (HA) solutions, was developed. The processing conditions to fabricate scaffolds with a layer-by-layer approach were optimized by studying their influence on fibres morphology and alignment. Two different scaffold architectures were designed and fabricated by tuning inter-fibre distance and fibres staggering. The developed scaffolds showed good reproducibility of the internal architecture characterized by highly porous, aligned fibres with an average diameter in the range 200-250 μm. Mechanical characterization showed that the architecture and HA loading influenced the scaffold compressive modulus and strength. Cell culture experiments employing MC3T3-E1 preosteoblast cell line showed good cell adhesion, proliferation, alkaline phosphatase activity and bone mineralization on the developed scaffolds.

  2. Culturing primary human osteoblasts on electrospun poly(lactic-co-glycolic acid) and poly(lactic-co-glycolic acid)/nanohydroxyapatite scaffolds for bone tissue engineering.

    Science.gov (United States)

    Li, Mengmeng; Liu, Wenwen; Sun, Jiashu; Xianyu, Yunlei; Wang, Jidong; Zhang, Wei; Zheng, Wenfu; Huang, Deyong; Di, Shiyu; Long, Yun-Ze; Jiang, Xingyu

    2013-07-10

    In this work, we fabricated polymeric fibrous scaffolds for bone tissue engineering using primary human osteoblasts (HOB) as the model cell. By employing one simple approach, electrospinning, we produced poly(lactic-co-glycolic acid) (PLGA) scaffolds with different topographies including microspheres, beaded fibers, and uniform fibers, as well as the PLGA/nanohydroxyapatite (nano-HA) composite scaffold. The bone-bonding ability of electrospun scaffolds was investigated by using simulated body fluid (SBF) solution, and the nano-HA in PLGA/nano-HA composite scaffold can significantly enhance the formation of the bonelike apatites. Furthermore, we carried out in vitro experiments to test the performance of electrospun scaffolds by utilizing both mouse preosteoblast cell line (MC 3T3 E1) and HOB. Results including cell viability, alkaline phosphatase (ALP) activity, and osteocalcin concentration demonstrated that the PLGA/nano-HA fibers can promote the proliferation of HOB efficiently, indicating that it is a promising scaffold for human bone repair.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    , and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation...... in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival...... of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from...

  4. FGF Suppresses Poldip2 Expression in Osteoblasts.

    Science.gov (United States)

    Katsumura, Sakie; Izu, Yayoi; Yamada, Takayuki; Griendling, Kathy; Harada, Kiyoshi; Noda, Masaki; Ezura, Yoichi

    2016-12-05

    Osteoporosis is one of the most prevalent ageing-associated diseases that are soaring in the modern world. Although various aspects of the disease have been investigated to understand the bases of osteoporosis, the pathophysiological mechanisms underlying bone loss is still incompletely understood. Poldip2 is a molecule that has been shown to be involved in cell migration of vascular cells and angiogenesis. However, expression of Poldip2 and its regulation in bone cells were not known. Therefore, we examined the Poldip2 mRNA expression and the effects of bone regulators on the Poldip2 expression in osteoblasts. We found that Poldip2 mRNA is expressed in osteoblastic MC3T3-E1 cells. As FGF controls osteoblasts and angiogenesis, FGF regulation was investigated in these cells. FGF suppressed the expression of Poldip2 in MC3T3-E1 cells in a time dependent manner. Protein synthesis inhibitor but not transcription inhibitor reduced the FGF effects on Poldip2 gene expression in MC3T3-E1 cells. As for bone-related hormones, dexamethasone was found to enhance the expression of Poldip2 in osteoblastic MC3T3-E1 cells whereas FGF still suppressed such dexamethasone effects. With respect to function, knockdown of Poldip2 by siRNA suppressed the migration of MC3T3-E1 cells. Poldip2 was also expressed in the primary cultures of osteoblast-enriched cells and FGF also suppressed its expression. Finally, Poldip2 was expressed in femoral bone in vivo and its levels were increased in aged mice compared to young adult mice. These data indicate that Poldip2 is expressed in osteoblastic cells and is one of the targets of FGF. J. Cell. Biochem. 9999: 1-8, 2017. © 2016 Wiley Periodicals, Inc.

  5. miR-223 contributes to the AGE-promoted apoptosis via down-regulating insulin-like growth factor 1 receptor in osteoblasts.

    Science.gov (United States)

    Qin, Yi; Ye, Jichao; Wang, Peng; Gao, Liangbin; Wang, Suwei; Shen, Huiyong

    2016-01-01

    Advanced glycation end products (AGEs) have been confirmed to induce bone quality deterioration in diabetes mellitus (DM), and to associate with abnormal expression of miRNAs in DM patients or in vitro Recently, miRNAs have been recognized to mediate the onset or progression of DM. In the present study, we investigated the regulation on miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells, with real-time quantitative PCR assay. And then we examined the inhibition of insulin-like growth factor 1 receptor (IGF-1R) expression by miR-223, via targeting of the 3' UTR of IGF-1R with real-time quantitative PCR, western blotting and luciferase reporter assay. Then we explored the regulation of miR-223 and IGF-1R levels, via the lentivirus-mediated miR-223 inhibition and IGF-1R overexpression in the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It was demonstrated that AGE-BSA treatment with more than 100 μg/ml significantly up-regulated miR-223 level, whereas down-regulated IGF-1R level in MC3T3-E1 cells. And the up-regulated miR-223 down-regulated IGF-1R expression in both mRNA and protein levels, via targeting the 3' UTR of IGF-1R Moreover, though the AGE-BSA treatment promoted apoptosis in MC3T3-E1 cells, the IGF-1R overexpression or the miR-223 inhibition significantly attenuated the AGE-BSA-promoted apoptosis in MC3T3-E1 cells. In summary, our study recognized the promotion of miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells. The promoted miR-223 targeted IGF-1R and mediated the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It implies that miR-223 might be an effective therapeutic target to antagonize the AGE-induced damage to osteoblasts in DM.

  6. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lei [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wu, Zhong [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wang, Jianguang, E-mail: jianguangwang@163.com [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Jianguo, E-mail: gehujianguo68@163.com [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China)

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

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

    Directory of Open Access Journals (Sweden)

    Sebastian M. Bonk

    2015-07-01

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

  8. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  9. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  10. Activities of the Deriviatives of Chitin on the Osbeoblast Proliferation and the Effect on Bone Strength in Ovariectomized Rats

    Institute of Scientific and Technical Information of China (English)

    WEI Changzheng; LIU Wanshun; HAN Baoqin; DUAN Yixiang

    2006-01-01

    This study intends to examine the effects of different concentrations of four kinds of degradations of chitin:glucosamine (GLC), N-acetyl-D-glucosamine (NAG), chitooligosaccharide(COS), CM-chitooligosaccharide (CM-COS)-on the proliferation of MC3T3-E1 cell line cultured in vitro.Results suggest that all of the glucoses mentioned above promoted the proliferation of osteoblast, and various concentrations have different effects: the proliferation was remarkable 100 , 100 , 500 , 500 μg/mL ,respectively. Furthermore we choose the glucosamine as the material and study the effect on the bone strength in ovariectomized rats. The results showed that the middle does of glucosamine can significantly increase the bone strength of femur in ovariectomized rats.

  11. TGF-beta suppresses POEM expression through ERK1/2 and JNK in osteoblasts.

    Science.gov (United States)

    Miyazono, Agasa; Yamada, Atsushi; Morimura, Naoko; Takami, Masamichi; Suzuki, Dai; Kobayashi, Makoto; Tezuka, Ken-Ichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2007-11-13

    POEM, also called nephronectin, is an extracellular matrix protein that is considered to play a critical role as an adhesion molecule in the development and functioning of various tissues, such as kidneys and bones. In the present study, we examined the molecular mechanism of POEM gene expression, and found that transforming growth factor-beta (TGF-beta) strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3-E1. TGF-beta-induced decrease of POEM expression occurred in both time- and dose-dependent manners through the activation of TGF-beta receptor I and extracellular signal-regulated kinase/c-Jun N-terminal kinase pathways.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-15

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

  13. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    Science.gov (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  14. Biocompatibility of artificial bone based on vancomycin loaded mesoporous silica nanoparticles and calcium sulfate composites.

    Science.gov (United States)

    Gu, Jisheng; Wang, Teng; Fan, Guoxin; Ma, Junhua; Hu, Wei; Cai, Xiaobing

    2016-04-01

    The aim of this study was to evaluate the in vitro and in vivo biocompatibility of artificial bone based on vancomycin loaded mesoporous silica nanoparticles and calcium sulfate composites. In vitro cytotoxicity tests by cholecystokinin octapeptide (CCK-8) assay showed that the 5%Van-MSN-CaSO4 and Van-CaSO4 bone cements were cytocompatible for mouse osteoblastic cell line MC3T3-E1. The microscopic observation confirmed that MC3T3-E1cells incubated with Van-CaSO4 group and 5%Van-MSN-CaSO4 group exhibited clear spindle-shaped changes, volume increase and maturation, showing that these cements supported adhesion of osteoblastic cells on their surfaces. In addition, the measurement of alkaline phosphatase activity revealed the osteoconductive property of these biomaterials. In order to assess in vivo biocompatibility, synthesized cements were implanted into the distal femur of twelve adult male and female New Zealand rabbits. After implantation in artificial defects of the distal femur, 5%Van-MSN-CaSO4 and Van-CaSO4 bone cements did not damage the function of main organs of rabbits. In addition, the Van-MSN-CaSO4 composite allowed complete repair of bone defects with new bone formation 3 months after implantation. These results show potential application of Van-MSN-CaSO4 composites as bone graft materials for the treatment of open fracture in human due to its mechanical, osteoconductive and potential sustained drug release characteristics and the absence of adverse effects on the body.

  15. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  16. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.

    2010-01-01

    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  17. PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

    Science.gov (United States)

    Xu, Xiaolong; Qiu, Sujun; Zhang, Yuxian; Yin, Jie; Min, Shaoxiong

    2017-03-01

    Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

  18. Parathyroid hormone enhances fluid shear-induced [Ca2+]i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca2+ channels

    Science.gov (United States)

    Ryder, K. D.; Duncan, R. L.

    2001-01-01

    Osteoblasts respond to both fluid shear and parathyroid hormone (PTH) with a rapid increase in intracellular calcium concentration ([Ca2+]i). Because both stimuli modulate the kinetics of the mechanosensitive cation channel (MSCC), we postulated PTH would enhance the [Ca2+]i response to fluid shear by increasing the sensitivity of MSCCs. After a 3-minute preflow at 1 dyne/cm2, MC3T3-E1 cells were subjected to various levels of shear and changes in [Ca2+]i were assessed using Fura-2. Pretreatment with 50 nM bovine PTH(1-34) [bPTH(1-34)] significantly enhanced the shear magnitude-dependent increase in [Ca2+]i. Gadolinium (Gd3+), an MSCC blocker, significantly inhibited the mean peak [Ca2+]i response to shear and shear + bPTH(1-34). Nifedipine (Nif), an L-type voltage-sensitive Ca2+ channel (VSCC) blocker, also significantly reduced the [Ca2+]i response to shear + bPTH(1-34), but not to shear alone, suggesting VSCC activation plays an interactive role in the action of these stimuli together. Activation of either the protein kinase C (PKC) or protein kinase A (PKA) pathways with specific agonists indicated that PKC activation did not alter the Ca2+ response to shear, whereas PKA activation significantly increased the [Ca2+]i response to lower magnitudes of shear. bPTH(1-34), which activates both pathways, induced the greatest [Ca2+]i response at each level of shear, suggesting an interaction of these pathways in this response. These data indicate that PTH significantly enhances the [Ca2+]i response to shear primarily via PKA modulation of the MSCC and VSCC.

  19. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

  20. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  1. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  2. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Genetics Group Web site at http://www.nist.gov/mml/biochemical/genetics/index.cfm . Once the total number... methods for human cell line authentication the identity of a cell line need no longer be in doubt. NIST...

  3. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  4. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  5. pELNS-BMPs与pELNS-Wnt3a联合构建表达对成骨细胞系成骨效能的作用研究%Osteogenesis capacity of constructing pELNS-BMPs and pELNS-Wnt3a co-expression in osteoblasts

    Institute of Scientific and Technical Information of China (English)

    阮文东; 雪原; 宗亚琪; 孙超

    2016-01-01

    and Wnt3a,then integrate them into the genome of mouse embryonic osteoblast cell,MC3T3-E1,and to explore the capability of osteogenic differentiation of individual bone morphogenetic control factor and the effective approaches to further improve the capability of osteogenic differentiation.Methods The plasmid vectors of gene expression including BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a were constructed,which were identified through enzyme cutting and further confirmed through sequencing.After packing pELNS-BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a,mouse embryonic osteoblast cell,MC3T3-E 1 was transfected,and the transfection efficiency was confirmed by GFP fluorescence imaging.The expression level of Runx2 mRNA and the transfection efficiency of individual bone morphogenetic control factor was detected by Real time PCR.Eight groups of MC3T3-E1 were dual-gene co-transfected,and the transfection efficiency was verified by GFP fluorescence imaging.ELISA was adopted to detect the expression level of BGP and ALP in MC3T3-E1culture supernatants;Real time PCR was adopted to detect the expression level of Runx2 mRNA;Western blot was adopted to detect the expression level of protein of BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a.Thus,the effectiveness of osteogenic differentiation of dual-gene co-transfection were evaluated.Results The recombination of lentiviruses,pELNS-BMP-2,pELNS-BMP-4,pELNS-BMP-6,pELNS-BMP-7,pELNS-BMP-9 and pELNS-Wnt3a were successfully constructed.MC3T3-E1 was successfully transfected.The expression levels of Runx2 mRNA were:BMP-2 > BMP-4 > BMP-9 > BMP-7 > Wnt3a > BMP-6.Successful transfection of the dual-gene co-transfection of eight groups of MC3T3-E1 were verified by GFP fluorescence imaging.The expression level of Runx2 mRNA,the expression of BGP and ALP showed BMP-2 and BMP-7 co-transfection group was the most efficient in osteogenesis transfection.Western blot revealed thatthe protein expression of BMP-2,BMP-4,BMP-6,BMP-7,BMP-9 and Wnt3a

  6. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  7. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  8. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  9. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  10. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  11. Engineering biomaterial surfaces using nanoparticle assemblies: A new paradigm for modulating cell function

    Science.gov (United States)

    Lipski, Anna Marie

    Silica nanoparticles (NP) were investigated as a surface modification medium and their impact on cell function was studied. This work has demonstrated that NP assemblies are suitable for the surface modification of both metal and polymer substrates. Additionally, important surface parameters, such as nano-roughness, charge, and chemistry, can be imparted in a predictable manner. More importantly, by varying the NP size, nano-roughness of a surface can be varied independent of chemistry. Two terminally differentiated mammalian cell types, bovine aortic endothelial cells (BAEC) and murine calvarial osteoblast-like cells (MC3T3-E1), were used to probe the effects of nano-topography on cell proliferation, metabolic activity, spreading, cytoskeletal F-actin alignment, and focal adhesion recruitment. Furthermore, the influence of nano-topography on cell migration was studied using BAEC and human fetal osteoblasts (hFOB). The results suggested that surface nano-rugosity affects cell behavior at various levels and that these effects are cell type specific; however, some clear trends were discerned with respect to F-actin alignment and cell migration. In particular, presentation of nano-features resulted in enhancement of cytoskeletal F-actin alignment along the long axis of the cells in comparison to unmodified glass. With respect to cell migration, increased nano-roughness resulted in decreased migration rates for both BAEC and hFOB. Finally, the potential of nano-rugosity as a mediator of cell differentiation was investigated by following the lineage specific differentiation of human marrow-derived mesenchymal progenitor cells (MPC) on NP-modified 316L stainless steel and titanium substrates. It was observed that NP modification enhanced the differentiation of MPC into an osteogenic lineage and that rugosity appeared to be the dominant factor in directing this differentiation. Thus, coatings composed of silica NPs presented a new paradigm that may lend themselves to

  12. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  13. Effect of Mg-Zn alloy on integrin beta-1 expression in osteoblasts%镁锌合金对成骨细胞整合素β1表达的影响

    Institute of Scientific and Technical Information of China (English)

    张岩; 陶海荣; 何耀华; 蒋垚; 张绍翔; 张小农

    2009-01-01

    BACKGROUND: A novel biodegradable Mg-Zn alloy is designed which the density and the Young's modulus are proximal to human bone. At the same time, it depletes the toxicity of aluminium and rare earth element in commercial magnesium alloys. OBJECTIVE: To observe the effect of Mg-Zn alloy (Mg-6%Zn) on the integrin βi expression of preosteoblasts MC3T3-E1. DESIGN, TIME AND SETTING: A contrast study was performed at the Central Laboratory of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University between March and May 2008. MATERIALS: The Mg-6% Zn was prepared by School of Materials Science and Engineering of Shanghai Jiao Tong University, which the density was 1.82 g/cm~3 and the Young's modulus was nearly 44 GPa. Poly-L-lactic acid (PLLA) was used as the controls. MC3T3-E1 cells were provided by Chinese Academy of Science Type Culture Collection. METHODS: The cell attachment was observed after cultured with Mg-Zn and PLLA at 2, 24 and 48 hours under scanning electron microscope; the integrin β1 mRNA expression of MC3T3-E1 cultured with Mg-Zn and PLLA was estimated by real-time fluorescent quantitative polymerase chain reaction (real-time PCR) at days 1, 3, 6, 9,12 and 15 after culture. MAIN OUTCOME MEASURES: The MC3T3-E1 cells attachment on the material surface and the integrin β1 mRNA expression. RESULTS: MC3T3-E1 cell adhesion was better on the Mg-Zn alloy surfaces than on the PLLA surface; The integrin (31 mRNA of osteoblasts on Mg-Zn kept on expressing during experiment and increased with time (P 0.05). CONCLUSION: MC3T3-E1 cell adhesion is better on the Mg-Zn alloy surfaces than on the PLLA surface, but it is not mediated by inducing the integrin p1 mRNA expression.%背景:自主设计的可降解镁锌合金既保持了镁合金与人骨接近的密度和弹性模量,又排除了商业镁合金中铝和稀土元素的毒性.目的:观察新型可降解镁锌合金(Mg-6%Zn)对前成骨细胞MC3T3-E1细胞整合素β1表达的影响.设计、

  14. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  15. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans

    2016-01-01

    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  16. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

    2011-01-01

    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...... except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein......The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...

  17. Biomineralization of a Self-Assembled Extracellular Matrix for Bone Tissue Engineering

    OpenAIRE

    Meng, Yizhi; Qin, Yi-Xian; DiMasi, Elaine; Ba, Xiaolan; Rafailovich, Miriam; Pernodet, Nadine

    2008-01-01

    Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein-mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, sync...

  18. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

    Full Text Available Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus. To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

  19. RETINOIC ACID DOWN-REGULATES BONE MORPHOGENETIC PROTEIN 7 EXPRESSION IN RAT WITH CLEFT PALATE

    Institute of Scientific and Technical Information of China (English)

    Lei Guo; Yu-yan Zhao; Shi-liang Zhang; Kui Liu; Xiao-yu Gao

    2008-01-01

    Objective To evaluate the effects of retinoic acid (RA) on expression of bone morphogenetic protein 7 (BMP-7)in rat fetus with cleft palate, and the effects of RA on proliferation and apoptosis of osteoblasts. Methods All-trans RA (ATRA) was used to induce congenital cleft palate in Wistar rat. BMP-7 mRNA expres-sion in maxillary bone tissue of fetal rats was measured by Northern blotting analysis. Flow cytometry and MTT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells. BMP-7 mRNA and protein ex-pressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis.Results ATRA could induce cleft palate of rat fetus. The incidence rate of cleft palate induced by 100 mg/kg AT-RA (45.5%) was significantly higher than 50 mg/kg ATRA (12.5%, P<0.05). BMP-7 mRNA expression de-creased in maxillary bone tissue of rat fetus with cleft palate. MC-3T3-E1 cells proliferation treated with 1 × 10-6 mol/L ATRA decreased by 60%, the cell apoptosis increased by 2 times. BMP-7 mR.NA and protein levels in MC-3T3-E1cells treated with 1 × 10-6 mol/L ATRA decreased by 60% and 80%, respectively, compared with ATRA-untreated ceils (P<0.05).Conclusions BMP-7 may play an important role in embryonic palate development RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.

  20. Effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

    Objective:To explore effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Methods: MTT and flow cytometry were adopted for detecting the effect of exogenous leptin on cell cycle of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Results: Leptin with mass concentration (0 ng/mL, 5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could stimulate the growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29; exogenous leptin with mass concentration (5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could inhibit cell growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29 after 72 h; among which, inhibiting effects of cell line-SGC7901 and cell line-HT-29 were the most significant under the effect of exogenous leptin with mass concentration-200 ng/mL.Conclusion:Within a certain concentration and action time, exogenous leptin can stimulate the growth of gastric adenocarcinoma cell line and colon cancer cell line, and then promot the tumor cell proliferation and/or inhibit the tumor cell apoptosis.

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

    Science.gov (United States)

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

    2014-11-01

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

  2. Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures.

    Science.gov (United States)

    Saito, Akihiro; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Chikazu, Daichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2016-04-29

    It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

  3. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  4. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  5. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  6. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

  7. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  8. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  9. Mechanical strain promotes osteoblast ECM formation and improves its osteoinductive potential

    Directory of Open Access Journals (Sweden)

    Guo Yong

    2012-10-01

    Full Text Available Abstract Background The extracellular matrix (ECM provides a supportive microenvironment for cells, which is suitable as a tissue engineering scaffold. Mechanical stimulus plays a significant role in the fate of osteoblast, suggesting that it regulates ECM formation. Therefore, we investigated the influence of mechanical stimulus on ECM formation and bioactivity. Methods Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with mechanical tensile strain. After removing the cells, the ECMs coated on dishes were prepared. The ECM protein and calcium were assayed and MC3T3-E1 cells were re-seeded on the ECM-coated dishes to assess osteoinductive potential of the ECM. Results The cyclic tensile strain increased collagen, bone morphogenetic protein 2 (BMP-2, BMP-4, and calcium levels in the ECM. Compared with the ECM produced by unstrained osteoblasts, those of mechanically stimulated osteoblasts promoted alkaline phosphatase activity, elevated BMP-2 and osteopontin levels and mRNA levels of runt-related transcriptional factor 2 (Runx2 and osteocalcin (OCN, and increased secreted calcium of the re-seeded MC3T3-E1 cells. Conclusion Mechanical strain promoted ECM production of osteoblasts in vitro, increased BMP-2/4 levels, and improved osteoinductive potential of the ECM. This study provided a novel method to enhance bioactivity of bone ECM in vitro via mechanical strain to osteoblasts.

  10. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  11. Involvement of microRNAs in regulation of osteoblastic differentiation in mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hideharu Okamoto

    Full Text Available UNLABELLED: BACKGOROUND: MicroRNAs (miRNAs, which regulate biological processes by annealing to the 3'-untranslated region (3'-UTR of mRNAs to reduce protein synthesis, have been the subject of recent attention as a key regulatory factor in cell differentiation. The effects of some miRNAs during osteoblastic differentiation have been investigated in mesenchymal stem cells, however they still remains to be determined in pluripotent stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Bone morphogenic proteins (BMPs are potent activators of osteoblastic differentiation. In the present study, we profiled miRNAs during osteoblastic differentiation of mouse induced pluripotent stem (iPS cells by BMP-4, in which expression of important osteoblastic markers such as Rux2, osterix, osteopontin, osteocalcin, PTHR1 and RANKL were significantly increased. A miRNA array analysis revealed that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a were significantly downregulated. Interestingly, miR-124a and miR-181a directly target the transcription factors Dlx5 and Msx2, both of which were increased by about 80-and 30-fold, respectively. In addition, transfection of miR-124a and miR-181a into mouse osteo-progenitor MC3T3-E1 cells significantly reduced expression of Dlx5, Runx2, osteocalcin and ALP, and Msx2 and osteocalcin, respectively. Finally, transfection of the anti-miRNAs of these six miRNAs, which are predicted to target Dlx5 and Msx2, into mouse iPS cells resulted in a significant increase in several osteoblastic differentiation markers such as Rux2, Msx2 and osteopontin. CONCLUSIONS/SIGNIFICANCE: In the present study, we demonstrate that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a miRNAs, especially miR-124a and miR-181a, are important regulatory factors in osteoblastic differentiation of mouse iPS cells.

  12. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  13. Further characterization of the first seminoma cell line TCam-2.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2008-03-01

    Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

  14. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  15. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  16. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  17. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  18. Pilose antler peptide protects osteoblasts from inflammatory and oxidative injury through EGF/EGFR signaling.

    Science.gov (United States)

    Chunhui, Yang; Wenjun, Cai; Hui, Wen; Liquan, Sha; Changwei, Zhao; Tianzhu, Zhang; Wenhai, Zhao

    2017-02-16

    Epidermal growth factor (EGF)/EFG receptor (EGFR) signaling plays an important role in the osteoblastogenesis. The potential effects of pilose antler peptide (PAP) on osteoblast cell damages was investigated in our present study through EGF/EGFR signaling. In MC3T3-E1 osteoblastic cells, PAP treatment significantly inhibited the production of inflammatory cytokines by decreasing the levels of serum proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). PAP treatment also alleviated the oxidative responses as indicated by increased activities of catalase (SOD) and decreased levels of malondialdehyde (MDA). EGF inhibition, by siRNA knockdown, almost abolished PAP-induced osteoblast cytoprotection against inflammation and oxidant stress. Further, our results showed that PAP stimulated the nuclear erythroid factor 2-related factor 2 (Nrf2)2/heme oxygenase-1(HO-1) signaling, and inhibited the activation of uclear factor kappa B (NF-κB) pathway in MC3T3-E1 cells. On the other hand, EGF siRNA knockdown inhibited PAP-induced cytoprotection, which decreased the expression of Nrf-2, HO-1 and increased the level of p-NF-κBp65, p-IκBα in MC3T3-E1 cells. Thus, our research demonstrated that PAP protects osteoblasts from inflammatory and oxidative injury through EGF/EGFR signaling.

  19. The Role of MicroRNA, miR-24, and Its Target CHI3L1 in Osteomyelitis Caused by Staphylococcus aureus.

    Science.gov (United States)

    Jin, Tao; Lu, Yong; He, Qing Xiao; Wang, Hai; Li, Bing Fu; Zhu, Liang Yue; Xu, Qing Yong

    2015-12-01

    Osteomyelitis is a debilitating infectious disease of the bone which is predominantly caused by Staphylococcus aureus (S. aureus). MicroRNAs (miRNAs) have been shown to play a regulatory role in osteogenesis. In the present study, the expression levels of miRNAs proposed to potentially play a regulatory role in bone formation or differentiation (miR-24, miR-29b, miR-200a, miR-208, miR-322) were analyzed in the whole blood of patients with bacterial osteomyelitis or healthy controls, and in MC3T3-E1 cells infected with S. aureus by qRT-PCR. The expression of miR-24 was significantly down-regulated in osteomyelitis patients and S. aureus-infected MC3T3-E1 cells compared with the healthy controls or untreated control cells. Moreover, our results showed that S. aureus inhibited MC3T3-E1 cell proliferation, induced osteoblast apoptosis and prohibited bone formation and mineralization. We found that overexpression of miR-24 could reduce the effects of S. aureus, while inhibition of miR-24 intensified the effects. We also demonstrated that miR-24 suppressed the expression of chitinase 3-like 1 (CHI3L1) mRNA, thought to mediate multiple signaling pathways, by directly binding to the 3'-untranslated region.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-01

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

  1. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  2. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  3. JKT-1 is not a human seminoma cell line.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; van Gurp, Ruud J H L M; van Drunen, Ellen; Beverloo, H Berna; Lau, Yun-Fai Chris; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Hatakeyama, Shingo; Ohyama, Chikara; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-08-01

    The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-08

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

  5. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  6. Development of a cell line from Echinococcus granulosus germinal layer.

    Science.gov (United States)

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  7. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  8. Establishment and characterization of rat portal myofibroblast cell lines.

    Directory of Open Access Journals (Sweden)

    Michel Fausther

    Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

  9. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  10. Apoptotic effect of noscapine in breast cancer cell lines.

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-12

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

  12. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  13. Investigation of the selenium metabolism in cancer cell lines.

    Science.gov (United States)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-02-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  14. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  15. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  16. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  17. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Radford, I.R. [Peter MacCallum Cancer Institute, East Melbourne, VIC (Australia). Research Division

    1998-12-31

    Differences in the radiosensitivity of normal and cancerous cells could arise in various ways. Although there is no compelling data to support the view, the currently prevailing opinion is that differences in radiosensitivity are related to differences in some aspect of enzymatic DNA repair. A test of the importance of possible differences in enzymatic DNA repair in determining relative radiosensitivity would be to compare lethality in cells containing equivalent numbers of DNA lesions. Six cell lines were used in these studies: two Chinese hamster (CHO and V79) and a monkey (Vero) fibroblast-like line, a mouse melanoma line (B16-F1), and a rat (RUC-2) and a human (SQ-20B) carcinoma line. This group of cell lines displays a wide range of sensitivities to external beam low-LET radiation, ranging from the relatively radiosensitive B16-F1 and Vero lines through to the highly radioresistant RUC-2 line. However, it is important to note that none of the lines has a demonstrated defect in enzymatic DNA repair and that all appear to die by necrosis following a lethal radiation insult. Despite having significantly different radiosensitivities, CHO and V79 cells showed comparable responses to DNA-associated {sup 125}I-decays with D{sub o} values of around 65. More surprisingly, the radiosensitive B16-F1 line and the radioresistant RUC-2 line both had responses with D{sub o} values of around 133 {sup 125}I-decays. The factor of two difference between the D{sub o} values for these two pairs of cell lines is probably attributable to CHO and V79 cells being pseudo-diploid whereas B 16-F1 and RUC2 appear to have derived from tetraploid cells. The generality of the above result, for DNA lesions of different quality, was tested by comparing the sensitivities of CHO and V79 cells to DNA-associated {sup 3}H-decays. Again, consistent with the {sup 125}I-decay data, there was no significant difference in the D{sub o} values for these lines. Our {sup 3}H- and {sup 125}I-decay data are

  18. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  19. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

  20. Establishment of a new bovine leukosis virus producing cell line.

    Science.gov (United States)

    Beier, D; Riebe, R; Blankenstein, P; Starick, E; Bondzio, A; Marquardt, O

    2004-11-01

    Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

  1. Expressional patterns of chaperones in ten human tumor cell lines

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    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  2. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    Science.gov (United States)

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  3. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  4. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  5. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

    OGINO, AKIYOSHI; SANO, EMIKO; OCHIAI, YUSHI; YAMAMURO, SHUN; TASHIRO, SHINYA; YACHI, KAZUNARI; OHTA, TAKASHI; FUKUSHIMA, TAKAO; OKAMOTO, YUTAKA; TSUMOTO, KOUHEI; UEDA, TAKUYA; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2014-01-01

    Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant

  6. Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

    Full Text Available Abstract Background Taurolidine (TRD represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas were incubated with increasing concentrations of TRD (100 μM, 250 μM and 1000 μM for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining. Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

  7. Exometabolom analysis of breast cancer cell lines: Metabolic signature.

    Science.gov (United States)

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-08-21

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

  8. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  9. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid.

  10. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  11. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  12. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  13. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

  14. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  15. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  16. Guidelines for the use of cell lines in biomedical research.

    Science.gov (United States)

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-09

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

  17. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

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

    Directory of Open Access Journals (Sweden)

    Guofeng Qian

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

  19. Reconstruction of endometrium from human endometrial side population cell lines.

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    Irene Cervelló

    Full Text Available Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC population recently identified by several groups using the side population (SP technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP cell lines (ICE 1-7: four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3 and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN. Phenotype analysis corroborated their epithelial (CD9+ or stromal (vimentin+ cell origin and mesenchymal (CD90+, CD73+ and CD45⁻ attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα or progesterone receptor (PR. The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

  20. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  1. Monoclonal antibodies against the human leukemia cell line K 562.

    Science.gov (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  2. 马来酸酐改性聚乳酸对成骨细胞黏附、增殖和分化的研究%Effects of Maleic Anhydride-modified Poly(D,L-lactic acid) on the Adhesion,Proliferation and Differentiation of Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    向燕; 王远亮; 罗彦凤; 张兵兵; 辛娟; 郑丹芳

    2011-01-01

    The main objective of this study was to observe the adhesion, proliferation and differentiation of mouse os-teblast-like MC3T3-E1 cells cultured on maleic anhydride-modified poly(D,L-lactic acid) (MPLA) and poly(D,L-lactic acid) (PDLLA) polymers, and to evaluate the cytocompatibility of MPLA polymer. The effects of MPLA and PDLLA polymers on the morphology, adhesion, proliferation, the content of total cellular protein, alkaline phospha-tase (ALP) activity and the content of Ca of MC3T3-E1 cells were explored. These results indicated that MC3T3-E1 cells on MPLA polymer adhered and spread more fully. On MPLA polymer, the proliferation, total protein content, ALP activity, Ca content of the cells were significantly higher than those of the cells on PDLLA polymer (P< 0. 01). It was concluded that MPLA polymer could promote the adhesion, spreading, proliferation and the synthesis of protein of osteoblasts, and also induced the differentiation and mineralization of osteoblasts, suggesting that MPLA polymer might have the better cytocompatibility than PDLLA.%通过研究马来酸酐改性聚乳酸(MPLA)和聚乳酸(PDLLA)材料表面对MC3T3-E1成骨细胞形态、黏附、增殖、细胞总蛋白含量、碱性磷酸酶活性及细胞分泌无机钙含量的影响,评价MPLA和PDLLA材料的细胞相容性.结果显示:与PDLLA相比,MPLA材料上的成骨细胞完全黏附和充分铺展;MPLA材料上细胞的增殖速率、细胞总蛋白含量、细胞碱性磷酸酶活性及细胞分泌的无机钙含量都显著高于PDLLA(P<0.01).这些结果说明,MPLA材料能促进MC3T3-E1成骨细胞的黏附、铺展、增殖及蛋白质的合成,并能促进成骨细胞的分化和矿化,与PDLLA材料相比具有更好的细胞相容性.

  3. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  4. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

    OpenAIRE

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-01-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members o...

  5. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  6. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

    Science.gov (United States)

    Meeth, Katrina; Wang, Jake Xiao; Micevic, Goran; Damsky, William; Bosenberg, Marcus W

    2016-09-01

    The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

  7. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  8. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  9. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  10. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  11. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

    Full Text Available Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2 and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS, mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  12. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  13. Development of a hybrid scaffold with synthetic biomaterials and hydrogel using solid freeform fabrication technology

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Jin-Hyung; Park, Min; Park, Jaesung; Cho, Dong-Woo [Department of Mechanical Engineering, POSTECH (Korea, Republic of); Kim, Jong Young, E-mail: dwcho@postech.ac.kr [Department of Mechanical Engineering, Andong National University (Korea, Republic of)

    2011-09-15

    Natural biomaterials such as hyaluronic acid, gelatin and collagen provide excellent environments for tissue regeneration. Furthermore, gel-state natural biomaterials are advantageous for encapsulating cells and growth factors. In cell printing technology, hydrogel which contains cells was printed directly to form three-dimensional (3D) structures for tissue or organ regeneration using various types of printers. However, maintaining the 3D shape of the printed structure, which is made only of the hydrogel, is very difficult due to its weak mechanical properties. In this study, we developed a hybrid scaffold consisting of synthetic biomaterials and natural hydrogel using a multi-head deposition system, which is useful in solid freeform fabrication technology. The hydrogel was intentionally infused into the space between the lines of a synthetic biomaterial-based scaffold. The cellular efficacy of the hybrid scaffold was validated using rat primary hepatocytes and a mouse pre-osteoblast MC3T3-E1 cell line. In addition, the collagen hydrogel, which encapsulates cells, was dispensed and the viability of the cells observed. We demonstrated superior effects of the hybrid scaffold on cell adhesion and proliferation and showed the high viability of dispensed cells.

  14. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    Science.gov (United States)

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  15. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  16. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  17. Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Valentina Pozzi

    2015-05-01

    Full Text Available Background/Aims: Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs, has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT, an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

  18. Boldine: a potential new antiproliferative drug against glioma cell lines.

    Science.gov (United States)

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent.

  19. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  20. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  1. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies.

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  2. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yonezawa, Takayuki [Department of Nutriproteomics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Lee, Ji-Won [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Hibino, Ayaka; Asai, Midori [Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Hojo, Hironori [Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Cha, Byung-Yoon [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Teruya, Toshiaki [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213 (Japan); Nagai, Kazuo [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Chung, Ung-Il [Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Yagasaki, Kazumi [Department of Nutriproteomics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Division of Applied Biological Chemistry, Institute of Agriculture, Tokyo Noko University, 3-5-8 Saiwai, Fuchu, Tokyo 183-8509 (Japan); and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  3. Osteogenic and antimicrobial nanoparticulate calcium phosphate and poly-(D,L-lactide-co-glycolide) powders for the treatment of osteomyelitis

    Energy Technology Data Exchange (ETDEWEB)

    Uskoković, Vuk, E-mail: vuk21@yahoo.com [Therapeutic Micro and Nanotechnology Laboratory, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA (United States); Hoover, Charles [Department of Cell and Tissue Biology, University of California, San Francisco, CA (United States); Vukomanović, Marija [Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Belgrade (Serbia); Advanced Materials Department, Jožef Stefan Institute, Ljubljana (Slovenia); Uskoković, Dragan P. [Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Belgrade (Serbia); Desai, Tejal A. [Therapeutic Micro and Nanotechnology Laboratory, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA (United States)

    2013-08-01

    Development of a material for simultaneous sustained and localized delivery of antibiotics and induction of spontaneous regeneration of hard tissues affected by osteomyelitis stands for an important clinical need. In this work, a comparative analysis of the bacterial and osteoblastic cell response to two different nanoparticulate carriers of clindamycin, an antibiotic commonly prescribed in the treatment of bone infection, one composed of calcium phosphate and the other comprising poly-(D,L-lactide-co-glycolide)-coated calcium phosphate, was carried out. Three different non-cytotoxic phases of calcium phosphate, exhibiting dissolution and drug release profiles in the range of one week to two months to one year, respectively, were included in the analysis: monetite, amorphous calcium phosphate and hydroxyapatite. Spherical morphologies and narrow size distribution of both types of nanopowders were confirmed in transmission and scanning electron microscopic analyses. The antibiotic-containing powders exhibited sustained drug release contingent upon the degradation rate of the carrier. Assessment of the antibacterial performance of the antibiotic-encapsulated powders against Staphylococcus aureus, the most common pathogen isolated from infected bone, yielded satisfactory results both in broths and on blood agar plates for all the analyzed powders. In contrast, no cytotoxic behavior was detected upon the incubation of the antibiotic powders with the osteoblastic MC3T3-E1 cell line for up to three weeks. The cells were shown to engage in a close contact with the antibiotic-containing particles, irrespective of their internal or surface phase composition, polymeric or mineral. At the same time, both types of particles upregulated the expression of osteogenic markers osteocalcin, osteopontin, Runx2 and protocollagen type I, suggesting their ability to promote osteogenesis and enhance remineralization of the infected site in addition to eliminating the bacterial source of

  4. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  5. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  6. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

    2012-09-01

    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  7. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  8. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  9. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

    de Jong, WK; ten Hacken, NHT; Groen, HJM

    2006-01-01

    Efficacy of third-line chemotherapy treatment for small cell lung cancer (SCLC) is unknown. We present our experience with third-tine chemotherapy for recurrent SCLC. Between January 1996 and July 2004 all. consecutive patients treated for SCLC were retrospectively studied. We recorded patient chara

  10. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S

    2007-01-01

    UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...

  11. Silicon Carbide Tiles for Sidewall Lining in Aluminium Electrolysis Cells

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

    The paper introduces the nitride bonded silicon carbide used for sidewall lining in aluminium eletrolysis cells ,including technical process,main properties and application results.Comparison tests on various physical properties of silicon carbide products made by LIRR and other producers worldwide have also been conducted in an independent laboratory.

  12. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  13. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  14. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  15. THP-1 cell line: an in vitro cell model for immune-modulation approach : Review

    NARCIS (Netherlands)

    Chanput, W.; Mes, J.J.; Wichers, H.J.

    2014-01-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review a

  16. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  17. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

    Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC50: 11 μM; 48 h IC50: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC50: 47 μM; 48 h IC50: 17 μM). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7-123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.

  18. Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line.

    Science.gov (United States)

    Gao, Shaorong; McGarry, Michelle; Ferrier, Tricia; Pallante, Benedetta; Priddle, Helen; Gasparrini, Bianca; Fletcher, Judy; Harkness, Linda; De Sousa, Paul; McWhir, Jim; Wilmut, Ian

    2003-02-01

    Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

  19. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  20. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  1. Centrosomal dysregulation in human metastatic melanoma cell lines.

    Science.gov (United States)

    Charters, Geoffrey A; Stones, Clare J; Shelling, Andrew N; Baguley, Bruce C; Finlay, Graeme J

    2011-09-01

    Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.

  2. BRITER: a BMP responsive osteoblast reporter cell line.

    Directory of Open Access Journals (Sweden)

    Prem Swaroop Yadav

    Full Text Available BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line, responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance.

  3. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Goestring, Lovisa [Affibody AB, Bromma (Sweden); Palm, Stig [Sahlgrenska Academy at Goeteborg University, Department of Radiation Physics, Goeteborg (Sweden); Carlsson, Joergen [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Rudbeck Laboratory, Biomedical Radiation Sciences, Uppsala (Sweden)

    2008-06-15

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin {sup registered} treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from {sup 211}At decays using the HER2-binding affibody molecule {sup 211}At-(Z{sub HER2:4}){sub 2} as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of {sup 211}At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from {sup 211}At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  4. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  5. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  6. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  7. Highly efficient site-specific transgenesis in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

    Full Text Available Abstract Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate

  8. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  9. Correlation between Twist expression and multidrug resistance of breast cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Yue-Xi Wang; Xiao-Mei Chen; Jun Yan; Zhi-Ping Li

    2016-01-01

    Objective:To study the correlation between Twist expression and multidrug resistance of breast cancer cell lines. Methods:Human breast cancer cell lines MCF-7, cisplatin-resistant human breast cancer cell lines MCF-7/DDP, doxorubicin-resistant human breast cancer cell lines MCF-7/Adr and taxol-resistant human breast cancer cell lines MCF/PTX were cultured, Twist in human breast cancer cell lines MCF-7 was overexpressed and treated with doxorubicin, and then cell viability and expression levels of EMT marker molecules and related signaling pathway molecules were detected. Results:mRNA contents and protein contents of Twist in drug-resistant breast cancer cell lines MCF-7/DDP, MCF-7/Adr and MCF/PTX were higher than those in MCF-7 cell lines;after doxorubicin treatment, inhibitory rates of cell viability in MCF-7 cell lines were higher than those in MCF-7/Adr and MCF-7/Twist cell lines;E-cadherin expression levels in MCF-7/Adr cell lines and MCF-7/Twist cell lines were lower than those in MCF-7 cell lines, and mRNA contents and protein contents of N-cadherin, Vimentin, TGF-β, Smad, Wnt,β-catenin, TNF-αand NF-kB were higher than those in MCF-7 cell lines. Conclusion:Increased expression of Twist is associated with the occurrence of drug resistance in breast cancer cells.

  10. "Helicobacter Pylori Attachment To 7 Mamalian Cell Lines "

    Directory of Open Access Journals (Sweden)

    N. Rahimi-Fard

    2006-06-01

    Full Text Available Background and Aim: Helicobacter pylori is the etiologic agent of chronic –active gastritis, gastroduodenal ulcers in humans, and a co-factor in the occurrence of gastric cancer and mucosa-associated lymphoid tumors, Adhesion of H.pylori to the gastric mucosa is a critical and also initial step in the pathogenesis of the disease. Bacterial adhesion inhibitory agents provide a novel pharmacologic approach to the management of infectious diseases. Materials and Methods: 22 H. pylori strains, isolated from the antral biopsies of 49 patients with dyspepsia, gastritis, gastric ulcer, duodenal ulcer,…were assayed by ELISA (UPRto investigate the diversity of attachment to 7 mamalian cell lines. Results: The concentration of H.pylori and cell suspention ,the condition and temperature, can alter the attachment rate.Best bacterial concentration was equal to 1 Mc farland,and for cell suspension was 5*10 cells/ml.90 minutes in 37C incubation period result in maximum attachment. H.pylori can attach to all 7 cell lines, there are no significant differences between 22 H.pylori strains in attachment to cells. The attachment pattern of H.pylori to the cells showed significant reduction respectly from HepII, HeLa, SW742, AGS,HT29/219, HT29 to Caco-2.Maximum attachment were seen to HepII, HeLa and SW742 cells, and among these HepII was the best cells for this purpose. Conclusion: Our studies suggest that Hep II, HeLa and SW742 cells could serve as a suitable in-vitro model for the study of H.pylori adhesions, attachment, inhibition of attachment and detachment assays and among these Hep II cell is prefer recommended.

  11. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B

    1999-01-01

    for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  12. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Science.gov (United States)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  13. Cytolytic replication of echoviruses in colon cancer cell lines

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    Gullberg Maria

    2011-10-01

    Full Text Available Abstract Background Colorectal cancer is one of the most common cancers in the world, killing nearly 50% of patients afflicted. Though progress is being made within surgery and other complementary treatments, there is still need for new and more effective treatments. Oncolytic virotherapy, meaning that a cancer is cured by viral infection, is a promising field for finding new and improved treatments. We have investigated the oncolytic potential of several low-pathogenic echoviruses with rare clinical occurrence. Echoviruses are members of the enterovirus genus within the family Picornaviridae. Methods Six colon cancer cell lines (CaCo-2, HT29, LoVo, SW480, SW620 and T84 were infected by the human enterovirus B species echovirus 12, 15, 17, 26 and 29, and cytopathic effects as well as viral replication efficacy were investigated. Infectivity was also tested in spheroids grown from HT29 cells. Results Echovirus 12, 17, 26 and 29 replicated efficiently in almost all cell lines and were considered highly cytolytic. The infectivity of these four viruses was further evaluated in artificial tumors (spheroids, where it was found that echovirus 12, 17 and 26 easily infected the spheroids. Conclusions We have found that echovirus 12, 17 and 26 have potential as oncolytic agents against colon cancer, by comparing the cytolytic capacity of five low-pathogenic echoviruses in six colon cancer cell lines and in artificial tumors.

  14. Genetic instability of cell lines derived from a single human small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Engelholm, S A; Vindeløv, L L; Spang-Thomsen, M

    1985-01-01

    Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations with differ......Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations...

  15. Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...

  16. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

  17. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  18. The cellular protooncogenes c-fos and egr-1 are regulated by prostacyclin in rodent osteoblasts and fibroblasts.

    Science.gov (United States)

    Glantschnig, H; Varga, F; Klaushofer, K

    1996-11-01

    PGs are local regulators of various cellular functions. They exert their effects via specific PG receptor subtypes. Induction of c-fos gene expression has been described for arachidonic acid and its metabolite PGE2. We demonstrate that another very short half-lifed prostanoid metabolite, namely prostacyclin (PGI2), is a regulator of immediate-early genes. PGI2 transiently induced the growth-associated immediate-early genes c-fos and egr-1 in osteoblastic as well as fibroblastic cell lines. Furthermore, we showed that PGI2 dose dependently stimulated new DNA synthesis in the osteoblastic cell line MC3T3-E1. Although PGI2 is known to be a potent inducer of cyclooxygenases, we showed that this pathway is not necessary for protooncogene induction by PGI2. Our data indicate a direct effect of PGI2 on immediate-early gene expression, which does not depend on the synthesis of other prostanoids. Intracellular signal transduction mechanisms were studied with the protein kinase inhibitor H-7, a potent inhibitor of PGI2-induced c-fos expression. Experiments with phorbol esters revealed that protein kinase C activity is not obligatory for the effect of PGI2 on c-fos expression. We conclude from these results that PGI2, a rapidly inactivated prostanoid, has a major impact on cellular oncogene expression and growth in mesenchymally derived cells.

  19. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expres-sion, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  20. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  1. Apoptosis in Raji cell line induced by influenza A virus

    Institute of Scientific and Technical Information of China (English)

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远

    2003-01-01

    Objective To study the apoptotic effects of influenza A virus on the Raji cell line. Methods Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.Results Raji cells infected with influenza A virus showed changes of morphology apoptotis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.Conclusions Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.

  2. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  3. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

    Directory of Open Access Journals (Sweden)

    Edyta Biskup

    2010-05-01

    Full Text Available We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa, Sézary syndrome (SeAx, and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK. Mac1 and Mac2a had the highest growth rate (doubling time 18-28 h, >90% cycling cells whereas SeAx was proliferating slowly (doub-ling time 55 h, approximately 35% cycling cells. Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma.

  4. Mistaken identity of widely used esophageal adenocarcinoma cell line TE-7.

    Science.gov (United States)

    Boonstra, Jurjen J; van der Velden, Albertina W; Beerens, Erwin C W; van Marion, Ronald; Morita-Fujimura, Yuiko; Matsui, Yasuhisa; Nishihira, Tetsuro; Tselepis, Chris; Hainaut, Pierre; Lowe, Anson W; Beverloo, Berna H; van Dekken, Herman; Tilanus, Hugo W; Dinjens, Winand N M

    2007-09-01

    Cancer of the esophagus is the seventh leading cause of cancer death worldwide. Esophageal carcinoma cell lines are useful models to study the biological and genetic alterations in these tumors. An important prerequisite of cell line research is the authenticity of the used cell lines because the mistaken identity of a cell line may lead to invalid conclusions. Estimates indicate that up to 36% of the cell lines are of a different origin or species than supposed. The TE series, established in late 1970s and early 1980s by Nishihira et al. in Japan, is one of the first esophageal cancer cell line series that was used throughout the world. Fourteen TE cell lines were derived from human esophageal squamous cell carcinomas and one, TE-7, was derived from a primary esophageal adenocarcinoma. In numerous studies, this TE-7 cell line was used as a model for esophageal adenocarcinoma because it is one of the few esophageal adenocarcinoma cell lines existing. We investigated the authenticity of the esophageal adenocarcinoma cell line TE-7 by xenografting, short tandem repeat profiling, mutation analyses, and array-comparative genomic hybridization and showed that cell line TE-7 shared the same genotype as the esophageal squamous cell carcinoma cell lines TE-2, TE-3, TE-12, and TE-13. In addition, for more than a decade, independent TE-7 cultures from Japan, United States, United Kingdom, France, and the Netherlands had the same genotype. Examination of the TE-7 cell line xenograft revealed the histology of a squamous cell carcinoma. We conclude that the TE-7 cell line, used in several laboratories throughout the world, is not an adenocarcinoma, but a squamous cell carcinoma cell line. Furthermore, the cell lines TE-2, TE-3, TE-7, TE-12, and TE-13 should be regarded as one single squamous cell carcinoma cell line.

  5. Establishment and characterization of primary lung cancer cell lines from Chinese population

    Institute of Scientific and Technical Information of China (English)

    Chao ZHENG; Yi-hua SUN; Xiao-lei YE; Hai-quan CHEN; Hong-bin JI

    2011-01-01

    Aim: To establish and characterize primary lung cancer cell lines from Chinese population.Methods: Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5%FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice.Results: Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations.Conclusion: These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

  6. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

    Science.gov (United States)

    Simcox, Amanda; Mitra, Sayan; Truesdell, Sharon; Paul, Litty; Chen, Ting; Butchar, Jonathan P; Justiniano, Steven

    2008-08-01

    Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

  7. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

    Directory of Open Access Journals (Sweden)

    Amanda Simcox

    2008-08-01

    Full Text Available Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12 (a constitutively activated form of Ras profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

  8. Apoptosis induced by propolis in human hepatocellular carcinoma cell line.

    Science.gov (United States)

    Choi, Y H; Lee, W Y; Nam, S Y; Choi, K C; Park, Y E

    1999-07-01

    Propolis has been reported to exhibit a wide spectrum of activities including antibiotic, antiviral, anti-inflammatory, immunostimulatory and tumor carcinostatic properties. We showed propolis induced apoptosis in a human hepatoma cell line (SNU449) by FITC-Annexin V/PI staining. We also compared the apoptosis inducing effect between Korean and Commercial (Sigma # p-1010) propolis. There was no difference on apoptosis between them.

  9. Effect of histone deacetylase inhibitor on proliferation of biliary tract cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Ning Xu; Xin Wang; Sheng-Quan Zou

    2008-01-01

    AIM: To explore the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the growth of biliary tract cancer cell lines (gallbladder carcinoma cell line and cholangiocarcinoma cell line) in v/vo and in vitro,and to investigate the perspective of histone deacetylase inhibitor in its clinical application.METHODS: The survival rates of gallbladder carcinoma cell line (Mz-ChA-I cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazoy tetrazolium (MTT) assay.A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-I cell line)was successfully established, and changes in the growth of transplanted tumor after treated with TSAwere measured.RESULTS: TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-I cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) in a dose-dependent manner.After the nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-I cell line) was successfully established, the growth of cancer was inhibited in the model, after treated with TSA.CONCLUSION: TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo.

  10. Pseudoislet of hybrid cellular spheroids from commercial cell lines.

    Science.gov (United States)

    Jo, Y H; Nam, B M; Kim, B Y; Nemeno, J G; Lee, S; Yeo, J E; Yang, W; Park, S H; Kim, Y S; Lee, J I

    2013-10-01

    Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 μm. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy.

  11. Propranolol induced chromosomal aberrations in Chinese hamster ovary cell line

    Directory of Open Access Journals (Sweden)

    Mozhgan Sedigh-Ardekani

    2013-03-01

    Full Text Available Propranolol (PL, a non-selective beta-blocker, is a cardiovascular drug widely used to treat hypertension. The present study was concerned with assessing the cytogenetic effects of this drug on Chinese hamster ovary (CHO cell line. MTT assay was then carried out to determine the cytotoxicity index (IC50 of the drug. The IC50 value of PL was 0.43±0.02 mM. To investigate the clastogenic effects of the drug, chromatid and chromosome breaks and polyploidy in metaphases were analyzed. CHO cells were exposed to different concentrations of the drug (0.1, 0.2, 0.3, 0.4 mM for 24 hours. Considering that PL has liver metabolism, experiments were carried out in the presence and absence of the metabolic activation system (S9 mix. Mitomycin-C and sodium arsenite were used as positive controls. It was observed that in cells treated with different PL concentrations as 0.1, 0.2 and 0.3 mM, the frequency of chromatid and chromosome breaks as well as polyploidy increased when compared with untreated CHO cells. The addition of S9 mix significantly decreased the chromatid breaks, chromosome breaks and polyploidy compared to the treatment of PL alone. It is concluded that, PL causes chromatid and chromosome aberrations in CHO cell line and the metabolic activation system (S9 mix, playing an important role in drug cytotoxicity reduction.

  12. Off-line test of the KISS gas cell

    Energy Technology Data Exchange (ETDEWEB)

    Hirayama, Yoshikazu, E-mail: yoshikazu.hirayama@kek.jp [Institute of Particle and Nuclear Studies (IPNS), High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Watanabe, Yutaka; Imai, Nobuaki; Ishiyama, Hironobu; Jeong, Sun-Chan; Miyatake, Hiroari; Oyaizu, Michihiro [Institute of Particle and Nuclear Studies (IPNS), High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Kim, Yung Hee [Seoul National University, Seoul 151 742 (Korea, Republic of); Mukai, Momo [Tsukuba University, Ibaraki 305 0006 (Japan); Matsuo, Yukari; Sonoda, Tetsu; Wada, Michiharu [Nishina Center for Accelerator-Based Science, RIKEN, Wako, Saitama 351 0198 (Japan); Huyse, Mark; Kudryavtsev, Yuri; Van Duppen, Piet [Instituut voor Kern-en Stralingsfysica, KU Leuven, B-3001 Leuven (Belgium)

    2013-12-15

    Highlights: • Construction of the KEK Isotope Separation System (KISS) at RIKEN. • Ionization scheme of an iron. • Measurement of transport time profile in a gas cell. -- Abstract: The KEK Isotope Separation System (KISS) has been constructed at RIKEN to study the β-decay properties of neutron-rich isotopes with neutron numbers around N = 126 for application to astrophysics. A key component of KISS is a gas cell filled with argon gas at a pressure of 50 kPa to stop and collect the unstable nuclei, where the isotopes of interest will be selectively ionized using laser resonance ionization. We have performed off-line tests to study the basic properties of the gas cell and of KISS using nickel and iron filaments placed in the gas cell.

  13. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

    DEFF Research Database (Denmark)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M;

    1998-01-01

    analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...... of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential...

  14. Generation, isolation, and maintenance of rodent mast cells and mast cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko;

    2006-01-01

    Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

  15. Additive manufacturing of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] scaffolds for engineered bone development.

    Science.gov (United States)

    Mota, Carlos; Wang, Shen-Yu; Puppi, Dario; Gazzarri, Matteo; Migone, Chiara; Chiellini, Federica; Chen, Guo-Qiang; Chiellini, Emo

    2017-01-01

    A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue-engineering scaffolds. In this study, the manufacture of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer-controlled wet-spinning system, was investigated. By optimizing the processing parameters, three-dimensional scaffolds with different internal architectures were fabricated, based on a layer-by-layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79-88%, fibre diameter 47-76 µm and pore size 123-789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase-inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell-culture experiments employing the MC3T3-E1 murine pre-osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright © 2014 John Wiley & Sons, Ltd.

  16. Beta-type calcium phosphates with and without magnesium: From hydrolysis of brushite powder to robocasting of periodic scaffolds.

    Science.gov (United States)

    Richard, Raquel C; Sader, Márcia S; Dai, Jisen; Thiré, Rossana M S M; Soares, Gloria D A

    2014-10-01

    Several approaches have attempted to replace extensive bone loss, but each of them has their limitation. Nowadays, additive manufacture techniques have shown great potential for bone engineering. The objective of this study was to synthesize beta tricalcium phosphate (β-TCP), beta tricalcium phosphate substituted by magnesium (β-TCMP), and biphasic calcium phosphate substituted by magnesium (BCMP) via hydrolysis and produce scaffolds for bone regeneration using robocasting technology. Calcium deficient apatites, with and without magnesium were obtained by hydrolysis, calcined and physico-chemically characterized. Colorimetric cell viability assay, calcium nodule formation, and the expression of alkaline phosphatase, osteocalcin, transforming growth factor beta-1 and collagen were assessed using a mouse osteoblastic cell line (MC3T3-E1). Direct-write assembly of cylindrical periodic scaffolds was done via robotic deposition using β-TCP, β-TCMP, and BCMP colloidal inks. The sintered scaffolds were characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, Archimede's method, and uniaxial compression test. According to the cell viability assay, the powders induced cell proliferation. Calcium nodule formation and bone markers activity suggested that the materials present potential value in bone tissue engineering. The scaffolds built by robocasting presented interconnected porous and exhibited mean compressive strength between 7.63 and 18.67 MPa, compatible with trabecular bone.

  17. Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

    Science.gov (United States)

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G

    2002-05-01

    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

  18. Differences in proliferation, differentiation, and cytokine production by bone cells seeded on titanium-nitride and cobalt-chromium-molybdenum surfaces.

    Science.gov (United States)

    van Hove, Ruud P; Nolte, Peter A; Semeins, Cornelis M; Klein-Nulend, Jenneke

    2013-08-01

    Titanium-nitride coating is used to improve cobalt-chromium-molybdenum implant survival in total knee arthroplasty, but its effect on osteoconduction is unknown. Chromium and cobalt ions negatively affect the growth and metabolism of cultured osteoblasts while enhancing osteoclastogenic cytokine production. Therefore, it was hypothesized that a titanium-nitride surface would enhance osteoblast proliferation and/or differentiation and reduce osteoclastogenic cytokine production compared with a cobalt-chromium-molybdenum surface. MC3T3-E1 osteoblasts showed increased proliferation and decreased differentiation on titanium-nitride, while cytokine interleukin-6 production was higher on porous cobalt-chromium-molybdenum (p cobalt-chromium-molybdenum surface.

  19. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  20. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    Science.gov (United States)

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...