3D polylactide-based scaffolds for studying human hepatocarcinoma processes in vitro

NASA Astrophysics Data System (ADS)

Scaffaro, Roberto; Lo Re, Giada; Rigogliuso, Salvatrice; Ghersi, Giulio

2012-08-01

We evaluated the combination of leaching techniques and melt blending of polymers and particles for the preparation of highly interconnected three-dimensional polymeric porous scaffolds for in vitro studies of human hepatocarcinoma processes. More specifically, sodium chloride and poly(ethylene glycol) (PEG) were used as water-soluble porogens to form porous and solvent-free poly(L,D-lactide) (PLA)-based scaffolds. Several characterization techniques, including porosimetry, image analysis and thermogravimetry, were combined to improve the reliability of measurements and mapping of the size, distribution and microarchitecture of pores. We also investigated the effect of processing, in PLA-based blends, on the simultaneous bulk/surface modifications and pore architectures in the scaffolds, and assessed the effects on human hepatocarcinoma viability and cell adhesion. The influence of PEG molecular weight on the scaffold morphology and cell viability and adhesion were also investigated. Morphological studies indicated that it was possible to obtain scaffolds with well-interconnected pores of assorted sizes. The analysis confirmed that SK-Hep1 cells adhered well to the polymeric support and emitted surface protrusions necessary to grow and differentiate three-dimensional systems. PEGs with higher molecular weight showed the best results in terms of cell adhesion and viability.

Long-term stability of dental adhesive incorporated by boron nitride nanotubes.

PubMed

Degrazia, Felipe Weidenbach; Leitune, Vicente Castelo Branco; Visioli, Fernanda; Samuel, Susana Maria Werner; Collares, Fabrício Mezzomo

2018-03-01

The aim of this study was to evaluate physicochemical properties, long-term microtensile bond strength and cytotoxicity of methacrylate-based adhesive containing boron nitride nanotubes (BNNTs) as fillers. A dental adhesive was formulated using BisGMA/HEMA, 66/33wt% (control). Inorganic BNNT fillers were incorporated into the adhesive at different concentrations (0.05, 0.075, 0.1 and 0.15wt%). Analyses of degree of conversion (DC), polymerization rate [Rp.(s -1 )], contact angle (CA) on dentin, after 24h and 6 months microtensile bond strength (μTBS-24h and 6 months) were assessed. Cytotoxicity was performed through viability of fibroblast cells (%) by sulforhodamine B (SRB) colorimetry. DC and max. polymerization rate increased (p<0.05) after incorporating 0.075 and 0.1wt% BNNT. The contact angle on dentin increased (p<0.05) after incorporating 0.15wt% BNNT. The μTBS-24h showed no changes (p>0.05) after incorporating up to 0.15wt% BNNT comparing to control. After 6 months, μTBS decreased (p<0.05) for control and 0.15wt% BNNT and BNNT groups up to 0.15wt% showed higher μTBS than control (p<0.05). No difference of fibroblast growth was found among adhesives (p>0.05) and up to 19% of cell viability was found comparing 0.05wt% BNNT to positive control group (100%). Incorporating boron nitride nanotubes up to 0.1wt% into dental adhesive increased the long-term stability to dentin without decreasing viability of fibroblast cell growth. Thus, the use of BNNTs as filler may decrease failure rate of current dentinal adhesives. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Cytocompatibility of polyethylene grafted with triethylenetetramine functionalized carbon nanoparticles

NASA Astrophysics Data System (ADS)

Žáková, Pavlína; Slepičková Kasálková, Nikola; Slepička, Petr; Kolská, Zdeňka; Karpíšková, Jana; Stibor, Ivan; Švorčík, Václav

2017-11-01

Various carbon nanostructures are widely researched as scaffolds for tissue engineering. We evaluated the surface properties and cell-substrate interactions of carbon nanoparticles functionalized with triethylenetetramine (CNPs) grafted polymer film. Two forms of polyethylene (HDPE, LDPE) were treated in an inert argon plasma discharge and, subsequently, grafted with CNPs. The surface properties were studied using multiple methods, including Raman spectroscopy, goniometry, atomic force microscopy, X-ray photoelectron spectroscopy and electrokinetic analysis. Cell-substrate interactions were determined in vitro by studying adhesion, proliferation and viability of vascular smooth muscle cells (VSMCs) from the aorta of a rat. Cell-substrate interactions on pristine and modified substrates were compared to standard tissue culture polystyrene. Our results show that CNPs affect surface morphology and wettability and therefore adhesion, proliferation and viability of cultured muscle cells.

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

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

In vitro electrochemical corrosion and cell viability studies on nickel-free stainless steel orthopedic implants.

PubMed

Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J; Rad, Armin Tahmasbi; Madihally, Sundararajan V; Tayebi, Lobat

2013-01-01

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments.

Use of novel chitosan hydrogels for chemical tissue bonding of autologous chondral transplants.

PubMed

Gittens, Jamila; Haleem, Amgad M; Grenier, Stephanie; Smyth, Niall A; Hannon, Charles P; Ross, Keir A; Torzilli, Peter A; Kennedy, John G

2016-07-01

The objective of this study was to evaluate the effect of chemical tissue bonding (CTB) on adhesion strength, fluid permeability, and cell viability across a cartilaginous graft-host interface in an in vitro autologous chondral transplant (ACT) model. Chitosan-based cross-linkers; Chitosan-Rose Bengal [Chi-RB (Ch-ABC)], Chitosan-Genipin [Chi-GP (Ch-ABC)], and Chitosan-Rose Bengal-Genipin [Chi-RB-GP (Ch-ABC)] were applied to bovine immature cartilage explants after pre-treatment with surface degrading enzyme, Chondroitinase-ABC (Ch-ABC). Adhesion strength, fluid permeability and cell viability were assessed via mechanical push-out shear testing, fluid transport and live/dead cell staining, respectively. All three chitosan-based cross-linkers significantly increased the adhesion strength at the graft-host interface, however, only a statistically significant decrease in fluid permeability was noted in Chi-GP (Ch-ABC) specimen compared to untreated controls. Cell viability was maintained for 7 days of culture across all three treatment groups. These results show the potential clinical relevance of novel chitosan-based hydrogels in enhancing tissue integration and reducing synovial fluid penetration after ACT procedures in diarthoidal joints such as the knee and ankle. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1139-1146, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

DPSC colonization of functionalized 3D textiles.

PubMed

Ortiz, Marine; Rosales-Ibáñez, Raúl; Pozos-Guillén, Amaury; De Bien, Charlotte; Toye, Dominique; Flores, Héctor; Grandfils, Christian

2017-05-01

Fiber scaffolds are attractive materials for mimicking, within a 3D in vitro system, any living environment in which animal cells can adhere and proliferate. In three dimensions, cells have the ability to communicate and organize into complex architectures similar to those found in their natural environments. The aim of this study was to evaluate, in terms of cell reactivity, a new in vitro cell model: dental pulp stem cells (DPSCs) in a 3D polymeric textile. Scaffolds were knitted from polyglycolic acid (PGA) or polydioxanone (PDO) fibers differing in surface roughness. To promote cell adhesion, these hydrophobic fabrics were also functionalized with either chitosan or the peptide arginine-glycine-aspartic acid (RGD). Cell behavior was examined 1, 10, and 21 days post-seeding with a LIVE/DEAD ® Kit. Confocal laser scanning microscopy (CLSM) highlighted the biocompatibility of these materials (cell survival rate: 94% to 100%). Fiber roughness was found to influence cell adhesion and viability significantly and favorably. A clear benefit of polymeric textile functionalization with chitosan or RGD was demonstrated in terms of cell adhesion and viability. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 785-794, 2017. © 2016 Wiley Periodicals, Inc.

In Vitro Cytotoxicity Assessment of an Orthodontic Composite Containing Titanium-dioxide Nano-particles.

PubMed

Heravi, Farzin; Ramezani, Mohammad; Poosti, Maryam; Hosseini, Mohsen; Shajiei, Arezoo; Ahrari, Farzaneh

2013-01-01

Background and aims. Incorporation of nano-particles to orthodontic bonding systems has been considered to prevent enamel demineralization around appliances. This study investigated cytotoxicity of Transbond XT adhesive containing 1 wt% titanium dioxide (TiO2) nano-particles. Materials and methods. Ten composite disks were prepared from each of the conventional and TiO2-containg composites and aged for 1, 3, 5, 7 and 14 days in Dulbecco's Modified Eagle's Medium (DMEM). The extracts were obtained and exposed to culture media of human gingival fibroblasts (HGF) and mouse L929 fibroblasts. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results. Both adhesives were moderately toxic for HGF cells on the first day of the experiment, but the TiO2-containing adhesive produced significantly lower toxicity than the pure adhesive (P<0.05). No significant differences were found in cell viability percentages between the two groups on the other days (P>0.05). There was a significant reduction in cell toxicity with increasing pre-incubation time (P<0.001). L929 cells showed similar toxicity trends, but lower sensitivity to detect cytotoxicity of dental composites. Conclusion. The orthodontic adhesive containing TiO2 nano-particles indicated comparable or even lower toxicity than its nano-particle-free counterpart, indicating that incorporation of 1 wt% TiO2 nano-particles to the composite structure does not result in additional health hazards compared to that occurring with the pure adhesive.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells.

PubMed

Silva, Mariana C C; de Paula, Cláudia A A; Ferreira, Joana G; Paredes-Gamero, Edgar J; Vaz, Angela M S F; Sampaio, Misako U; Correia, Maria Tereza S; Oliva, Maria Luiza V

2014-07-01

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Photoinitiator-Free Synthesis of Endothelial Cell Adhesive and Enzymatically Degradable Hydrogels

PubMed Central

Jones, Derek R.; Marchant, Roger E.; von Recum, Horst; Gupta, Anirban Sen; Kottke-Marchant, Kandice

2015-01-01

We report on a photoinitiator-free synthetic method of incorporating bioactivity into poly(ethylene glycol) (PEG) hydrogels in order to control physical properties, enzymatic biodegradability and cell-specific adhesiveness of the polymer network, while eliminating the need for UV-mediated photopolymerization. To accomplish this, hydrogel networks were polymerized using Michael addition with four-arm PEG acrylate (10 kDa), using a collagenase sensitive peptide (CSP) as a crosslinker, and introducing an endothelial cell adhesive peptide either terminally (RGD) or attached to the crosslinking peptide sequence (CSP-RGD). The efficiency of the Michael addition reactions were determined by NMR and Ellman’s assay. Successful decoupling of cell adhesivity and physical properties was demonstrated by quantifying and comparing the swelling ratios and Young’s Moduli of various hydrogel formulations. Degradation profiles were established by incubating functionalized hydrogels in collagenase solutions (0.0 – 1.0 µg/mL), demonstrating that functionalized hydrogels degraded at a rate dependent upon collagenase concentration. Moreover, it was shown that the degradation rate was independent of CSP-RGD concentration. Cell attachment and proliferation on functionalized hydrogels were compared for various RGD concentrations, providing evidence that cell attachment and proliferation were directly related to relative amounts of the CSP-RGD combination peptide. An increase in cell viability was achieved using Michael addition techniques when compared to UV-polymerization, and was assessed by a LIVE/DEAD fluorescence assay. This photoinitiator-free method shows promise in creating hydrogel-based tissue engineering scaffolds allow for decoupled cell adhesivity and physical properties and that render greater cell viability. PMID:25462848

Use of arginine-glycine-aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft.

PubMed

Schussler, O; Coirault, C; Louis-Tisserand, M; Al-Chare, W; Oliviero, P; Menard, C; Michelot, R; Bochet, P; Salomon, D R; Chachques, J C; Carpentier, A; Lecarpentier, Y

2009-03-01

Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine-glycine-aspartic acid-serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds. Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month. The method used for the RGD+ cell-collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36 A, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation. We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol.

PubMed

Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

2017-01-01

The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol.

Efficacy of surface-generated nitric oxide against Candida albicans adhesion and biofilm formation.

PubMed

Privett, Benjamin J; Nutz, Steven T; Schoenfisch, Mark H

2010-11-01

This report details the efficacy of nitric oxide (NO)-releasing xerogel surfaces composed of N-(6-aminohexyl)aminopropyl trimethoxysilane (AHAP3) and isobutyltrimethoxysilane (BTMOS) against Candida albicans adhesion, viability, and biofilm formation. A parallel plate flow cell assay was used to examine the effect of NO on planktonic fungal cells. Nitric oxide fluxes as low as 14 pmol cm(-2) s(-1) were sufficient to reduce fungal adhesion by ∼49% over the controls after 90 min. By utilizing a fluorescence live/dead assay and replicate plating, NO flux was determined to reduce fungal viability in a dose-dependent manner. The formation of C. albicans biofilms on NO-releasing xerogel-coated silicon rubber (SiR) coupons was impeded when compared to control (non-NO-releasing) and bare SiR surfaces. The synergistic efficacy of NO and silver sulfadiazine against adhered fungal cells and biofilms is reported with increased killing and biofilm inhibition over NO alone.

Development of functional biointerfaces by surface modification of polydimethylsiloxane with bioactive chlorogenic acid.

PubMed

Wu, Ming; He, Jia; Ren, Xiao; Cai, Wen-Sheng; Fang, Yong-Chun; Feng, Xi-Zeng

2014-04-01

The effect of physicochemical surface properties and chemical structure on the attachment and viability of bacteria and mammalian cells has been extensively studied for the development of biologically relevant applications. In this study, we report a new approach that uses chlorogenic acid (CA) to modify the surface wettability, anti-bacterial activity and cell adhesion properties of polydimethylsiloxane (PDMS). The chemical structure of the surface was obtained by X-ray photoelectron spectroscopy (XPS), the roughness was measured by atomic force microscopy (AFM), and the water contact angle was evaluated for PDMS substrates both before and after CA modification. Molecular modelling showed that the modification was predominately driven by van der Waals and electrostatic interactions. The exposed quinic-acid moiety improved the hydrophilicity of CA-modified PDMS substrates. The adhesion and viability of E. coli and HeLa cells were investigated using fluorescence and phase contrast microscopy. Few viable bacterial cells were found on CA-coated PDMS surfaces compared with unmodified PDMS surfaces. Moreover, HeLa cells exhibited enhanced adhesion and increased spreading on the modified PDMS surface. Thus, CA-coated PDMS surfaces reduced the ratio of viable bacterial cells and increased the adhesion of HeLa cells. These results contribute to the purposeful design of anti-bacterial surfaces for medical device use. Copyright © 2013 Elsevier B.V. All rights reserved.

In Vitro Electrochemical Corrosion and Cell Viability Studies on Nickel-Free Stainless Steel Orthopedic Implants

PubMed Central

Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J.; Rad, Armin Tahmasbi; Madihally, Sundararajan V.; Tayebi, Lobat

2013-01-01

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments. PMID:23630603

In Vitro Cytotoxicity Assessment of an Orthodontic Composite Containing Titanium-dioxide Nano-particles

PubMed Central

Heravi, Farzin; Ramezani, Mohammad; Poosti, Maryam; Hosseini, Mohsen; Shajiei, Arezoo; Ahrari, Farzaneh

2013-01-01

Background and aims. Incorporation of nano-particles to orthodontic bonding systems has been considered to prevent enamel demineralization around appliances. This study investigated cytotoxicity of Transbond XT adhesive containing 1 wt% titanium dioxide (TiO2) nano-particles. Materials and methods. Ten composite disks were prepared from each of the conventional and TiO2-containg composites and aged for 1, 3, 5, 7 and 14 days in Dulbecco’s Modified Eagle’s Medium (DMEM). The extracts were obtained and exposed to culture media of human gingival fibroblasts (HGF) and mouse L929 fibroblasts. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results. Both adhesives were moderately toxic for HGF cells on the first day of the experiment, but the TiO2-containing adhesive produced significantly lower toxicity than the pure adhesive (P<0.05). No significant differences were found in cell viability percentages between the two groups on the other days (P>0.05). There was a significant reduction in cell toxicity with increasing pre-incubation time (P<0.001). L929 cells showed similar toxicity trends, but lower sensitivity to detect cytotoxicity of dental composites. Conclusion. The orthodontic adhesive containing TiO2 nano-particles indicated comparable or even lower toxicity than its nano-particle-free counterpart, indicating that incorporation of 1 wt% TiO2 nano-particles to the composite structure does not result in additional health hazards compared to that occurring with the pure adhesive. PMID:24578816

Voltage effects on cells cultured on metallic biomedical implants

NASA Astrophysics Data System (ADS)

Haerihosseini, Seyed Morteza

Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. Surface potential of biomedical implants and excursions from resting open circuit potential (OCP), which is the voltage they attain while in contact with an electrolyte, can significantly change the interfacial properties of the metallic surfaces and alter the behavior of the surrounding cells, compromising the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. To date, the details of the physico-chemical phenomena and the role of different biomaterial parameters involved in the interaction between cells and metallic surfaces under cathodic bias have not been fully elucidated. In this work, changes in the interfacial properties of a CoCrMo biomedical alloy (ASTM F-1537) in phosphate-buffered saline (PBS) (pH 7.4) at different voltages was studied. Step polarization impedance spectroscopy technique was used to apply 50 mV voltage steps to samples, and the time-based current transients were recorded. A new equation was derived based on capacitive discharge through a Tafel element and generalized to deal with non-ideal impedance behavior. The new function compared to the KWW-Randles function, better matched the time-transient response. The results also showed a voltage dependent oxide resistance and capacitance behavior. Additionally, the in-vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each voltage were measured and related to the observed responses. Results show that cathodic and anodic voltages outside the voltage viability range (-400 < V < +500) lead to primarily intrinsic apoptotic and necrotic cell death, respectively. Cell death is associated with cathodic current densities of 0.1 uAcm-2 and anodic current densities of 10 uAcm-2. Significant increase in metallic ions (Co, Cr, Ni, Mo) was seen at +500 mV, and -1000 mV (Cr only) compared to open circuit potential. The number and total projected area of adhesion complexes was also lower on the polarized alloy (p < 0.05). These results show that reduction reactions on CoCrMo alloys leads to apoptosis of cells on the surface and may be a relevant mode of cell death for metallic implants in-vivo. . On the other hand, we studied how surface oxide thickness of Ti affects its voltage viability range and cellular response and whether anodic oxidation can serve as a means to extend this range. Cellular behavior (cell viability, cytoskeletal organization, and cellular adhesion) on bare and anodized Ti samples, potentiostatically held at voltages at the cathodic edge of the viability range, were assessed. Surfaces were characterized using contact angle (CA) measurement technique and atomic force microscopy (AFM), and the observed cellular response was related to the changes in the electrochemical properties (electrochemical currents, open circuit potential, and impedance spectra) of the samples. Results show that anodization at a low voltage (9 V) in phosphate buffer saline (PBS) generates a compact surface oxide with comparable surface roughness and energy to the starting native oxide on the bare surface. The anodized surface extends the viability range at 24 hours by about a 100 mV in the cathodic region, and preserved the cytoskeletal integrity and cell adhesion. Broadening of the viability range corresponds to an increase in impedance of the anodized surface at -400 mV(Ag/AgCl) and the resulting low average currents (below 0.1 uAcm-2) at the interface, which diminish the harmful cathodic reactions. Finally, cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesions in transiently transfected MC3T3-E1 pre-osteoblasts cultured on a CoCrMo alloy polarized at the cathodic and anodic edges of its voltage viability range (-400 and +500 mV(Ag/AgCl) respectively) were studied. Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) was also studied on the polarized metal at -1000, -400, and +500 mV(Ag/AgCl). The results show that at -400 mV(Ag/AgCl) a gradual loss of adhesion occurs over 24 hours while cells shrink in size during this time. At +500 mV, cells become non-viable after 5 hours without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at -1000 mV decreased sharply within 15 minutes after electrochemical polarization, which rendered the cells completely non-viable. No significant amount of ROS was released by cells on the polarized CoCrMo at any of these voltages.

Emphasizing the role of surface chemistry on hydrophobicity and cell adhesion behavior of polydimethylsiloxane/TiO2 nanocomposite films.

PubMed

Yousefi, Seyedeh Zahra; Tabatabaei-Panah, Pardis-Sadat; Seyfi, Javad

2018-07-01

Improving the bioinertness of materials is of great importance for developing biomedical devices that contact human tissues. The main goal of this study was to establish correlations among surface morphology, roughness and chemistry with hydrophobicity and cell adhesion in polydimethylsiloxane (PDMS) nanocomposites loaded with titanium dioxide (TiO 2 ) nanoparticles. Firstly, wettability results showed that the nanocomposite loaded with 30 wt.% of TiO 2 exhibited a superhydrophobic behavior; however, the morphology and roughness analysis proved that there was no discernible difference between the surface structures of samples loaded with 20 and 30 wt.% of nanoparticles. Both cell culture and MTT assay experiments showed that, despite the similarity between the surface structures, the sample loaded with 30 wt.% nanoparticles exhibits the greatest reduction in the cell viability (80%) as compared with the pure PDMS film. According to the X-ray photoelectron spectroscopy results, the remarkable reduction in cell viability of the superhydrophobic sample could be majorly attributed to the role of surface chemistry. The obtained results emphasize the importance of adjusting the surface properties especially surface chemistry to gain the optimum cell adhesion behavior. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of Rebamipide, a Novel Antiulcer Agent, on Helicobacter pylori Adhesion to Gastric Epithelial Cells

PubMed Central

Hayashi, Shunji; Sugiyama, Toshiro; Amano, Ken-Ichi; Isogai, Hiroshi; Isogai, Emiko; Aihara, Miki; Kikuchi, Mikio; Asaka, Masahiro; Yokota, Kenji; Oguma, Keiji; Fujii, Nobuhiro; Hirai, Yoshikazu

1998-01-01

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. The adhesion of H. pylori to human gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is thus a therapeutic target in the prevention of H. pylori infection. Experiments were performed to evaluate the effect of rebamipide, a novel antiulcer agent, on H. pylori adhesion to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from human gastric carcinomas, were used as target cells. Ten H. pylori strains isolated from patients with chronic gastritis and gastric ulcer were used in the study. We evaluated the effect of rebamipide on H. pylori adhesion to MKN-28 and MKN-45 cells quantitatively using our previously established enzyme-linked immunosorbent assay. The adhesion of H. pylori to MKN-28 and MKN-45 cells was significantly inhibited by pretreatment of these cells with 100 μg of rebamipide per ml. However, the adhesion was not affected by the pretreatment of H. pylori with rebamipide. On the other hand, the viabilities of H. pylori, MKN-28 cells, and MKN-45 cells were not affected by rebamipide. Our studies suggest that rebamipide inhibits the adhesion of H. pylori to gastric epithelial cells. PMID:9687380

Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.

We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwovenmore » scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.« less

EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells.

PubMed

Galler, K M; Widbiller, M; Buchalla, W; Eidt, A; Hiller, K-A; Hoffer, P C; Schmalz, G

2016-06-01

To evaluate the effect of dentine conditioning on migration, adhesion and differentiation of dental pulp stem cells. Dentine discs prepared from extracted human molars were pre-treated with EDTA (10%), NaOCl (5.25%) or H2 O. Migration of dental pulp stem cells towards pre-treated dentine after 24 and 48 h was assessed in a modified Boyden chamber assay. Cell adhesion was evaluated indirectly by measuring cell viability. Expression of mineralization-associated genes (COL1A1, ALP, BSP, DSPP, RUNX2) in cells cultured on pre-treated dentine for 7 days was determined by RT-qPCR. Nonparametric statistical analysis was performed for cell migration and cell viability data to compare different groups and time-points (Mann-Whitney U-test, α = 0.05). Treatment of dentine with H2 O or EDTA allowed for cell attachment, which was prohibited by NaOCl with statistical significance (P = 0.000). Furthermore, EDTA conditioning induced cell migration towards dentine. The expression of mineralization-associated genes was increased in dental pulp cells cultured on dentine after EDTA conditioning compared to H2 O-pre-treated dentine discs. EDTA conditioning of dentine promoted the adhesion, migration and differentiation of dental pulp stem cells towards or onto dentine. A pre-treatment with EDTA as the final step of an irrigation protocol for regenerative endodontic procedures has the potential to act favourably on new tissue formation within the root canal. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

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

PubMed

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

2007-05-01

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

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

Cardiomyocytes In Vitro Adhesion Is Actively Influenced by Biomimetic Synthetic Peptides for Cardiac Tissue Engineering

PubMed Central

Huerta-Cantillo, Rocio; Comisso, Marina; Danesin, Roberta; Ghezzo, Francesca; Naso, Filippo; Gastaldello, Alessandra; Schittullo, Eleonora; Buratto, Edward; Spina, Michele; Gerosa, Gino; Dettin, Monica

2012-01-01

Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding. PMID:22011064

Comparison of Six Different Silicones In Vitro for Application as Glaucoma Drainage Device

PubMed Central

Windhövel, Claudia; Harder, Lisa; Bach, Jan-Peter; Teske, Michael; Grabow, Niels; Eickner, Thomas; Chichkov, Boris; Nolte, Ingo

2018-01-01

Silicones are widely used in medical applications. In ophthalmology, glaucoma drainage devices are utilized if conservative therapies are not applicable or have failed. Long-term success of these devices is limited by failure to control intraocular pressure due to fibrous encapsulation. Therefore, different medical approved silicones were tested in vitro for cell adhesion, cell proliferation and viability of human Sclera (hSF) and human Tenon fibroblasts (hTF). The silicones were analysed also depending on the sample preparation according to the manufacturer’s instructions. The surface quality was characterized with environmental scanning electron microscope (ESEM) and water contact angle measurements. All silicones showed homogeneous smooth and hydrophobic surfaces. Cell adhesion was significantly reduced on all silicones compared to the negative control. Proliferation index and cell viability were not influenced much. For development of a new glaucoma drainage device, the silicones Silbione LSR 4330 and Silbione LSR 4350, in this study, with low cell counts for hTF and low proliferation indices for hSF, and silicone Silastic MDX4-4210, with low cell counts for hSF and low proliferation indices for hTF, have shown the best results in vitro. Due to the high cell adhesion shown on Silicone LSR 40, 40,026, this material is unsuitable. PMID:29495462

Real-time, label-free monitoring of cell viability based on cell adhesion measurements with an atomic force microscope.

PubMed

Yang, Fang; Riedel, René; Del Pino, Pablo; Pelaz, Beatriz; Said, Alaa Hassan; Soliman, Mahmoud; Pinnapireddy, Shashank R; Feliu, Neus; Parak, Wolfgang J; Bakowsky, Udo; Hampp, Norbert

2017-03-22

The adhesion of cells to an oscillating cantilever sensitively influences the oscillation amplitude at a given frequency. Even early stages of cytotoxicity cause a change in the viscosity of the cell membrane and morphology, both affecting their adhesion to the cantilever. We present a generally applicable method for real-time, label free monitoring and fast-screening technique to assess early stages of cytotoxicity recorded in terms of loss of cell adhesion. We present data taken from gold nanoparticles of different sizes and surface coatings as well as some reference substances like ethanol, cadmium chloride, and staurosporine. Measurements were recorded with two different cell lines, HeLa and MCF7 cells. The results obtained from gold nanoparticles confirm earlier findings and attest the easiness and effectiveness of the method. The reported method allows to easily adapt virtually every AFM to screen and assess toxicity of compounds in terms of cell adhesion with little modifications as long as a flow cell is available. The sensitivity of the method is good enough indicating that even single cell analysis seems possible.

Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices.

PubMed

Nagahara, Yukitoshi; Sekine, Hiroaki; Otaki, Mari; Hayashi, Masakazu; Murase, Norio

2016-02-01

Animal cells are generally cryopreserved in cryovials in a cell suspension state containing 5%-10% v/v dimethyl sulfoxide (DMSO) used as a cryoprotective agent. However, cryopreservation of cells in an attached state has not been intensively studied, and the effective freezing solution remains unknown. Here we determined the suitable DMSO concentration for the cryopreservation of human hepatoma HepG2 cells attached to glass and polydimethylsiloxane (PDMS) matrices coated with poly-l-lysine. With the use of the glass matrix, the rate of cell adhesion increased with the DMSO concentration up to 30% v/v in the freezing solution. In contrast, the cell-adhesion rate remained constant in the case of the PDMS matrix irrespective of the DMSO concentration between 10% v/v and 30% v/v. The viability of post-thawed cells attached to glass or PDMS matrix was also investigated. The viability was highest at the DMSO concentration of 20% v/v in the freezing solution. The DMSO concentration of 30% v/v, however, had a cytotoxic effect on the cell viability. Thus, the 20% v/v DMSO concentration was found to be most suitable for the cryopreservation of HepG2 cells in the attached state. This dose is high compared to the DMSO concentration used for the cryopreservation of cells in the suspended state. Copyright © 2015 Elsevier Inc. All rights reserved.

Cellular proliferation, cellular viability, and biocompatibility of HA-ZnO composites.

PubMed

Saha, Naresh; Dubey, Ashutosh K; Basu, Bikramjit

2012-01-01

One of the important issues in the development of hydroxyapatite (HA)-based biomaterials is the prosthetic infection, which limits wider use of monolithic HA despite superior cellular response. Recently, we reported that ZnO addition to HA can induce bactericidal property. It is therefore important to assess how ZnO addition influences the cytotoxicity property and cell adhesion/proliferation on HA-ZnO composite surfaces in vitro. In the above perspective, the objective of this study is to investigate the cell type and material composition dependent cellular proliferation and viability of pressureless sintered HA-ZnO composites. The combination of cell viability data as well as morphological observations of cultured human osteoblast-like SaOS2 cells and mouse fibroblast L929 cells suggests that HA-ZnO composites containing 10 Wt % or lower ZnO exhibit the ability to support cell adhesion and proliferation. Both SaOS2 and L929 cells exhibit extensive multidirectional network of actin cytoskeleton and cell flattening on the lower ZnO containing (≤10 Wt %) HA-ZnO composites. The in vitro results illustrate how variation in ZnO content can influence significantly the cell vitality, as evaluated using MTT biochemical assay. Also, the critical statistical analysis reveals that ZnO addition needs to be carefully tailored to ensure good in vitro cytocompatibility. The underlying reasons for difference in biological properties are analyzed. It is suggested that surface wettability as well as dissolution of ZnO, both contribute to the observed differences in cellular viability and proliferation. Copyright © 2011 Wiley Periodicals, Inc.

In vitro dentin barrier cytotoxicity testing of some dental restorative materials.

PubMed

Jiang, R D; Lin, H; Zheng, G; Zhang, X M; Du, Q; Yang, M

2017-03-01

To investigate the cytotoxicity of four dental restorative materials in three-dimensional (3D) L929 cell cultures using a dentin barrier test. The cytotoxicities of light-cured glass ionomer cement (Vitrebond), total-etching adhesive (GLUMA Bond5), and two self-etching adhesives (GLUMA Self Etch and Single Bond Universal) were evaluated. The permeabilities of human dentin disks with thicknesses of 300, 500, and 1000μm were standardized using a hydraulic device. Test materials and controls were applied to the occlusal side of human dentin disks. The 3D-cell scaffolds were placed beneath the dentin disks. After a 24-h contact with the dentin barrier test device, cell viabilities were measured by performing MTT assays. Statistical analysis was performed using the Mann-Whitney U test. The mean (SD) permeabilities of the 300-μm, 500-μm, and 1000-μm dentin disks were 0.626 (0.214), 0.219 (0.0387) and 0.089 (0.028) μlmin -1 cm -2 cm H 2 O -1 . Vitrebond was severely cytotoxic, reducing the cell viability to 10% (300-μm disk), 17% (500μm), and 18% (1000μm). GLUMA Bond5 reduced the cell viability to 40% (300μm), 83% (500μm), and 86% (1000μm), showing moderate cytotoxicity (300-μm) and non-cytotoxicity (500-μm and 1000-μm). Single Bond Universal and GLUMA Self Etch did not significantly reduce cell viability, regardless of the dentin thicknesses, which characterized them as non-cytotoxic. Cytotoxicity varied with the materials tested and the thicknesses of the dentin disks. The tested cytotoxicity of materials applied on 300-, 500-, and 1000-μm dentin disks indicates that the clinical use of the test materials (excepting self-etching adhesives) in deep cavities poses a potential risk of damage to the pulp tissues to an extent, depending on the thickness of the remaining dentin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modulating macrophage response to biomaterials

NASA Astrophysics Data System (ADS)

Zaveri, Toral

Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Since foreign body response limits performance and functional life of numerous implanted biomaterials/medical devices, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate this response. In this work we have explored two independent approaches to modulate the macrophage inflammatory response to biomaterials. The first approach targets surface integrins, cell surface receptors that mediate cell adhesion to biomaterials through adhesive proteins spontaneously adsorbed on biomaterial surfaces. The second approach involves surface modification of biomaterials using nanotopographic features since nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. More specifically, Zinc Oxide (ZnO) nanorod surface was investigated for its role in modulating macrophage adhesion and survival in vitro and foreign body response in vivo. For the first approach, we have investigated the role of integrin Mac-1 and RGD-binding integrins in the in-vivo osteolysis response and macrophage inflammatory processes of phagocytosis as well as inflammatory cytokine secretion in response to particulate biomaterials. We have also investigated the in vivo foreign body response (FBR) to subcutaneously implanted biomaterials by evaluating the thickness of fibrous capsule formed around the implants after 2 weeks of implantation. The role of Mac-1 integrin was isolated using a Mac-1 KO mouse and comparing it to a WT control. The role of RGD binding integrins in FBR was investigated by coating the implanted biomaterial with ELVAX(TM) polymer loaded with Echistatin which contains the RGD sequence. For the in-vivo osteolysis study and to study the in-vitro macrophage response to particulate biomaterials, we used the RGD peptide encapsulated in ELVAX(TM) and dissolved in macrophage media respectively. By studying the phagocytosis, inflammatory and FBR of macrophages from integrin knockout mice, as well as using various integrin blocking techniques we aim to identify the role of various integrins in macrophage inflammatory response. These integrins can serve as therapeutic targets for mitigating this inflammatory response and improve functional life of implanted biomaterials. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. In order to determine long-term physiological responses, ZnO nanorodcoated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 days. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. Hence although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent. Considering the toxicity of ZnO nanorod surface towards macrophages, their role as an antibacterial surface was explored. Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant-associated infection. To assess the efficacy of ZnO nanorod surfaces as an anti-bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO and glass substrates. Common implant-associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. ZnO nanorod surface and sputtered ZnO demonstrated a significant bactericidal effect, killing respectively 2.5x and 1.7x times the number of bacteria dead on glass. A similar bactericidal effect of ZnO substrates on S. epidermidis was also evident, with sputtered ZnO and ZnO nanorod substrates killing respectively 22x and 32x times bacteria dead on glass. These data support the further investigation of ZnO nanorod coatings for bacterial adhesion resistance and bactericidal properties.

Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

PubMed

Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

2010-08-06

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. Copyright 2010 Elsevier Inc. All rights reserved.

Adhesion modification of neural stem cells induced by nanoscale ripple patterns

NASA Astrophysics Data System (ADS)

Pedraz, P.; Casado, S.; Rodriguez, V.; Giordano, M. C.; Buatier de Mongeot, F.; Ayuso-Sacido, A.; Gnecco, E.

2016-03-01

We have studied the influence of anisotropic nanopatterns (ripples) on the adhesion and morphology of mouse neural stem cells (C17.2) on glass substrates using cell viability assay, optical microscopy and atomic force microscopy. The ripples were produced by defocused ion beam sputtering with inert Ar ions, which physically remove atoms from the surface at the energy of 800 eV. The ripple periodicity (∼200 nm) is comparable to the thickness of the cytoplasmatic microspikes (filopodia) which link the stem cells to the substrate. All methods show that the cell adhesion is significantly lowered compared to the same type of cells on flat glass surfaces. Furthermore, the AFM analysis reveals that the filopodia tend to be trapped parallel or perpendicular to the ripples, which limits the spreading of the stem cell on the rippled substrate. This opens the perspective of controlling the micro-adhesion of stem cells and the orientation of their filopodia by tuning the anisotropic substrate morphology without chemical reactions occurring at the surface.

Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

PubMed Central

Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

2011-01-01

The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652

HMC05, Herbal Formula, Inhibits TNF-α-Induced Inflammatory Response in Human Umbilical Vein Endothelial Cells

PubMed Central

Lee, Jong Suk; Park, Su-Young; Thapa, Dinesh; Kim, Ah Ra; Shin, Heung-Mook; Kim, Jung-Ae

2011-01-01

Vascular inflammation has been implicated in the progression of cardiovascular diseases such as atherosclerosis. In the present study, we found that HMC05, an extract from eight different herbal mixtures, dose-dependently inhibited tumor necrosis factor-α (TNF-α)-induced adhesion of monocytes to endothelial cells. Such inhibitory effect of HMC05 correlated with suppressed expression of monocyte chemoattractant protein-1, CC chemokine receptor 2, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1. In addition, HMC05 significantly inhibited production of reactive oxygen species (ROS) and nuclear factor (NF)-κB activation by TNF-α. Those inhibitory effects of HMC05 (1–10 μg mL−1) on the TNF-α-induced inflammatory event was similar to those of berberine (1–10 μM), which is a major component of HMC05 and one of herbal compounds known to have vasorelaxing and lipid-lowering activities. However, berberine significantly reduced the viability of HUVECs in a time- and concentration-dependent manner. In contrast, HMC05 (1–10 μg ml−1) did not affect the cell viability for up to 48 h treatment. In conclusion, we propose that HMC05 may be a safe and potent herbal formula against vascular inflammation, and its action may be attributable to the inhibition of ROS- and NF-κB-dependent expression of adhesion molecules and chemokines. PMID:19736220

In vitro cellular adhesion and antimicrobial property of SiO2-MgO-Al2O3-K2O-B2O3-F glass ceramic.

PubMed

Kalmodia, Sushma; Molla, Atiar Rahaman; Basu, Bikramjit

2010-04-01

The aim of the present study was to examine the cellular functionality and antimicrobial properties of SiO(2)-MgO-Al(2)O(3)-K(2)O-B(2)O(3)-F glass ceramics (GC) containing fluorophlogopite as major crystalline phase. The cellular morphology and cell adhesion study using human osteoblast-like Saos-2 cells and mouse fibroblast L929 cells reveals good in vitro cytocompatibility of GC. The potential use of the GC for biomedical application was also assessed by in vitro synthesis of the alkaline phosphatase (ALP) activity of Saos-2 cells. It is proposed that B(2)O(3) actively enhances the cell adhesion and supports osteoconduction process, whereas, fluorine component significantly influences cell viability. The Saos-2 and L929 cells on GC shows extensive multidirectional network of actin cytoskeleton. The in vitro results of this study illustrate how small variation in fluorine and boron in base glass composition influences significantly the biocompatibility and antimicrobial bactericidal property, as evaluated using a range of biochemical assays. Importantly, it shows that the cell viability and osteoconduction can be promoted in glass ceramics with lower fluorine content. The underlying reasons for difference in biological properties are analyzed and reported. It is suggested that oriented crystalline morphology in the lowest fluorine containing glass ceramic enhanced cellular spreading. Overall, the in vitro cell adhesion, cell flattening, cytocompatibility and antimicrobial study of the three different compositions of glass ceramic clearly reveals that microstructure and base glass composition play an important role in enhancing the cellular functionality and antimicrobial property.

[Exploratory study on the micro-remodeling of dermal tissue].

PubMed

Jiang, Yu-zhi; Ding, Gui-fu; Lu, Shu-liang

2009-10-01

To explore the effect of three-dimensional structure of dermal matrix on biological behavior of fibroblasts (Fb) in the microcosmic perspective. The three-dimensional structure of dermal tissue was analyzed by plane geometric and trigonometric function. Microdots structure array with cell adhesion effect was designed by computer-assisted design software according to the adhesive and non-adhesive components of dermal tissue. Four sizes (8 microm x 3 microm, space 6 microm; 16 microm x 3 microm, space 6 microm; 16 microm x 5 microm, space 8 microm; 20 microm x 3 microm, space 2 microm) of micropier grid used for cell culture (MPGCC) with cell-adhesive microdots, built up with micro-pattern printing and molecule self-assembly method were used to culture dermal Fb. Fb cultured with cell culture matrix without micropier grid was set up as control. The expression of skeleton protein (alpha-SMA) of Fb, cell viability and cell secretion were detected with immunohistochemistry, fluorescent immunohistochemistry, MTT test and the hydroxyproline content assay. The three-dimensional structure of dermal tissue could be simulated by MPGCC as shown in arithmetic analysis. Compared with those of control group [(12 +/- 3)% and (0.53 +/- 0.03) microg/mg, (0.35 +/- 0.04)], the expression of alpha-SMA [(49 +/- 3)%, (61 +/- 3)%, (47 +/- 4)%, (51 +/- 3)%] and the content of hydroxyproline [(0.95 +/- 0.04), (0.87 +/- 0.03), (0.81 +/- 0.03), (0.77 +/- 0.03) microg/mg] were increased significantly (P < 0.05), the cell viability of Fb (0.12 +/- 0.03, 0.13 +/- 0.04, 0.14 +/- 0.03, 0.19 +/- 0.03) cultured in MPGCC was decreased significantly (P < 0.05). When the parameters of micropier grid were changed, the expression of alpha-SMA, the cell viability and the content of hydroxyproline of Fb cultured in four sizes of MPGCC were also significantly changed as compared with one another (P < 0.05). MPGCC may be the basic functional unit of dermal template, or unit of dermal template to call. Different three-dimensional circumstances for dermal tissue can result in different template effect and wound healing condition.

Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

NASA Astrophysics Data System (ADS)

Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

2017-04-01

Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

Differential Effect of Zoledronic Acid on Human Vascular Smooth Muscle Cells

PubMed Central

Albadawi, Hassan; Haurani, Mounir J.; Oklu, Rahmi; Trubiano, Jordan P.; Laub, Peter J.; Yoo, Hyung-Jin; Watkins, Michael T.

2012-01-01

Introduction The activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether Zoledronic Acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating vs. quiescent cells and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration. Methods Human aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly ADP Ribose polymerase (PARP) enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly ADP- ribosylated proteins (PAR) respectively. Results There was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating vs. quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of PARP activity. Conclusions These data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion and migration which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation. PMID:23164362

The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

PubMed

Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

2013-07-01

To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cytotoxicity of titanium and silicon dioxide nanoparticles

NASA Astrophysics Data System (ADS)

Wagner, Stefanie; Münzer, Simon; Behrens, Peter; Scheper, Thomas; Bahnemann, Detlef; Kasper, Cornelia

2009-05-01

Different TiO2 and SiO2 nanoparticles have been tested concerning their toxicity on selected mammalian cell lines. Various powders and suspensions, all of which consist of titanium or silicon dioxide nanoparticles have been examined. These particles differ in the crystal structure, the size and the BET-surface area. There was also a classification in fixed particles and in particles easily accessible in solution. With focus on the possible adsorption of the nanoparticles into the human organism, via skin and via respiratory tract, the effects on fibroblasts (NIH-3T3) and on a human lung adenocarcinoma epithelial cell line were examined. Additionally, the particles were tested with HEP-G2 cells, which are often used as model cell line for biocompatibility tests, and PC-12 cells, a rat adrenal pheochromocytoma cell line. The viability of the cells was examined by the MTT-test. The viability results were found to partly depend on the type of cells used. The experimental results show that the adhesion of the cells on the different powders strongly depends on the type of cell lines as well as on the type of powder. It was found that the lower viability of some cells on the powder coatings is not only caused by a cytotoxicity effect of the powders, but is also due to a lower adhesion of the cells on the particle surfaces. Furthermore, it could be shown that the physical properties of the powders cannot be easily correlated to any observed biological effect. While some powders show a significant suppression of the cell growth, others with similar physical properties indicate no toxic effect.

Mesenchymal stem cells cultured on magnetic nanowire substrates

NASA Astrophysics Data System (ADS)

Perez, Jose E.; Ravasi, Timothy; Kosel, Jürgen

2017-02-01

Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 μm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing hMSCs differentiation.

Evaluation of the biological tolerability of the starch-based medical device 4DryField® PH in vitro and in vivo a rat model.

PubMed

Poehnert, Daniel; Abbas, Mahmoud; Maegel, Lavinia; Sambale, Franziska; Lavrentieva, Antonina; Kreipe, Hans-Heinrich; Klempnauer, Jürgen; Winny, Markus

2015-10-01

To evaluate in vitro cytotoxicity/biocompatibility as well as in vivo tolerability of the novel polysaccharide 4DryField® PH, certified for haemostasis and adhesion prevention. In vitro cytotoxicity/viability testing according to ISO EN 10,993 using murine and human tumour cell lines incubated with 4DryField® PH (PlantTec Medical GmbH). Using a rat model the impact of 4DryField® PH on animals viability and in vivo effects were macro- and micropathologically assessed. In vitro testing revealed no cytotoxic effect of 4DryField® PH nor enhancement of viability to tumour cell lines. In vivo viability of rats was unimpaired by 4DryField® PH. Bodyweight loss in animals with abdominal injury plus treatment with 4DryField® PH was in the range of controls and less than in injured rats without treatment. At day 7 after surgery no formation of adhesions, neither macroscopic nor histological remnants nor signs of foreign body reaction were present in animals without injury. In animals with peritoneal injury and 4DryField® PH application, histopathological observation revealed minor residuals of polysaccharide in the depth of wound cavity embedded in a thickened subperitoneal layer; however, with a suggested intact neoperitoneum. The presence of mononuclear cells surrounding polysaccharide particles in varying states of degradation was observable as well. 4DryField® PH is not cytotoxic and does not enhance viability of tumour cell lines. High dose of 4DryField® PH of 1.09 g/kg bodyweight is well tolerated and reduces weight loss in animals with peritoneal injury. The biocompatibility of 4DryField® PH can be rated as being excellent. © The Author(s) 2015.

Agglomeration, sedimentation, and cellular toxicity of alumina nanoparticles in cell culture medium

NASA Astrophysics Data System (ADS)

Yoon, Dokyung; Woo, Daekwang; Kim, Jung Heon; Kim, Moon Ki; Kim, Taesung; Hwang, Eung-Soo; Baik, Seunghyun

2011-06-01

The cytotoxicity of alumina nanoparticles (NPs) was investigated for a wide range of concentration (25-200 μg/mL) and incubation time (0-72 h) using floating cells (THP-1) and adherent cells (J774A.1, A549, and 293). Alumina NPs were gradually agglomerated over time although a significant portion of sedimentation occurred at the early stage within 6 h. A decrease of the viability was found in floating (THP-1) and adherent (J774A.1 and A549) cells in a dose-dependent manner. However, the time-dependent decrease in cell viability was observed only in adherent cells (J774A.1 and A549), which is predominantly related with the sedimentation of alumina NPs in cell culture medium. The uptake of alumina NPs in macrophages and an increased cell-to-cell adhesion in adherent cells were observed. There was no significant change in the viability of 293 cells. This in vitro test suggests that the agglomeration and sedimentation of alumina NPs affected cellular viability depending on cell types such as monocytes (THP-1), macrophages (J774A.1), lung carcinoma cells (A549), and embryonic kidney cells (293).

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds.

PubMed

Inanç, Bülend; Arslan, Y Emre; Seker, Sükran; Elçin, A Eser; Elçin, Y Murat

2009-07-01

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.

Human dental pulp stem cell adhesion and detachment in polycaprolactone electrospun scaffolds under direct perfusion

PubMed Central

Paim, A.; Braghirolli, D.I.; Cardozo, N.S.M.; Pranke, P.; Tessaro, I.C.

2018-01-01

Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005–3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion. PMID:29590258

Upregulation of ADAM12 contributes to accelerated cell proliferation and cell adhesion-mediated drug resistance (CAM-DR) in Non-Hodgkin's Lymphoma.

PubMed

Yin, Haibing; Zhong, Fei; Ouyang, Yu; Wang, Qiru; Ding, Linlin; He, Song

2017-10-01

ADAM12 is a member of a disintegrin and metalloproteinase family and has been reported to participate in the development of variety of tumors. However, the role of ADAM12 in Non-Hodgkin Lymphoma (NHL) has not been investigated. The present study was undertaken to determine the expression and biologic function of ADAM12 in human NHL. First, we constructed a model of cell adhesion in NHL, the mRNA, and protein level of ADAM12 in suspension and the adhesion model was analyzed by RT-PCR and western blot. Then, flow cytometry assay and western blot were used to investigate the mechanism of ADAM12 in the proliferation of NHL cells. In vitro, after using siRNA interfering ADAM12 expression, we performed adhesion assay and cell viability assay to determine the effect of ADAM12 on adhesive rate and drug sensitivity. ADAM12 was lowly expressed in suspended cells and highly expressed in adherent NHL cells. In addition, ADAM12 was positively correlated with the proliferation and apoptosis of NHL cells by regulating the expression of p-AKT and p-GSK-3β. Furthermore, ADAM12 promoted cell adhesion-mediated drug resistance (CAM-DR) in DLBCL via AKT signaling pathway. Our data support a role for ADAM12 in NHL cell proliferation, adhesion, and drug resistance, and it may pave the way for a novel therapeutic approach for CAM-DR in NHL.

Zeolite inorganic scaffolds for novel biomedical application: Effect of physicochemical characteristic of zeolite membranes on cell adhesion and viability

NASA Astrophysics Data System (ADS)

Tavolaro, Palmira; Catalano, Silvia; Martino, Guglielmo; Tavolaro, Adalgisa

2016-09-01

The design, preparation and selection of inorganic materials useful as functional scaffolds for cell adhesion is a complex question based both on the understanding of the chemical behavior of the materials and individual cells, and on their interactions. Pure zeolite membranes formed from synthetic crystals offer chemically-capable being modulated silanolic surfaces that are amenable to adhesion and growth of fibroblasts. We report the facile preparation of reusable, very longlasting, biocompatible, easily sterilized synthetic scaffolds in a zeolite membrane configuration, which are very stable in aqueous media (apart from ionic strength and pH values), able to adsorb pollutant species and to confine undesired toxic ions (present in culture media). This may ultimately lead to the development of cell supports for economic antibiotic-free culture media.

Hydrogenated amorphous silicon coatings may modulate gingival cell response

NASA Astrophysics Data System (ADS)

Mussano, F.; Genova, T.; Laurenti, M.; Munaron, L.; Pirri, C. F.; Rivolo, P.; Carossa, S.; Mandracci, P.

2018-04-01

Silicon-based materials present a high potential for dental implant applications, since silicon has been proven necessary for the correct bone formation in animals and humans. Notably, the addition of silicon is effective to enhance the bioactivity of hydroxyapatite and other biomaterials. The present work aims to expand the knowledge of the role exerted by hydrogen in the biological interaction of silicon-based materials, comparing two hydrogenated amorphous silicon coatings, with different hydrogen content, as means to enhance soft tissue cell adhesion. To accomplish this task, the films were produced by plasma enhanced chemical vapor deposition (PECVD) on titanium substrates and their surface composition and hydrogen content were analyzed by means of X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectrophotometry (FTIR) respectively. The surface energy and roughness were measured through optical contact angle analysis (OCA) and high-resolution mechanical profilometry respectively. Coated surfaces showed a slightly lower roughness, compared to bare titanium samples, regardless of the hydrogen content. The early cell responses of human keratinocytes and fibroblasts were tested on the above mentioned surface modifications, in terms of cell adhesion, viability and morphometrical assessment. Films with lower hydrogen content were endowed with a surface energy comparable to the titanium surfaces. Films with higher hydrogen incorporation displayed a lower surface oxidation and a considerably lower surface energy, compared to the less hydrogenated samples. As regards mean cell area and focal adhesion density, both a-Si coatings influenced fibroblasts, but had no significant effects on keratinocytes. On the contrary, hydrogen-rich films increased manifolds the adhesion and viability of keratinocytes, but not of fibroblasts, suggesting a selective biological effect on these cells.

Adhesion of Asaia bogorensis to Glass and Polystyrene in the Presence of Cranberry Juice.

PubMed

Antolak, Hubert; Kregiel, Dorota; Czyzowska, Agata

2015-06-01

The aim of the study was to evaluate the adhesion abilities of the acetic acid bacterium Asaia bogorensis to glass and polystyrene in the presence of American cranberry (Vaccinium macrocarpon) juice. The strain of A. bogorensis used was isolated from spoiled commercial fruit-flavored drinking water. The cranberry juice was analyzed for polyphenols, organic acids, and carbohydrates using high-performance liquid chromatography and liquid chromatography-mass spectrometry techniques. The adhesive abilities of bacterial cells in culture medium supplemented with cranberry juice were determined using luminometry and microscopy. The viability of adhered and planktonic bacterial cells was determined by the plate count method, and the relative adhesion coefficient was calculated. This strain of A. bogorensis was characterized by strong adhesion properties that were dependent upon the type of surface. The highest level of cell adhesion was found on the polystyrene. However, in the presence of 10% cranberry juice, attachment of bacterial cells was three times lower. Chemical analysis of juice revealed the presence of sugars, organic acids, and anthocyanins, which were identified as galactosides, glucosides, and arabinosides of cyanidin and peonidin. A-type proanthocyanidins responsible for the antiadhesion properties of V. macrocarpon also were detected.

Tetrandrine suppresses lung cancer growth and induces apoptosis, potentially via the VEGF/HIF-1α/ICAM-1 signaling pathway

PubMed Central

Chen, Zhuo; Zhao, Liang; Zhao, Feng; Yang, Guanghai; Wang, Jian Jun

2018-01-01

The present study investigated the effect of tetrandrine on lung cancer cell growth and apoptosis, and its possible underlying molecular mechanism. A549 human lung cancer cells were incubated with between 2.5 and 10 µM tetrandrine for 12, 24 and 48 h, following which the effect of tetrandrine on cell viability and apoptosis were assessed using an MTT assay and flow cytometry. ELISA and western blotting were used to analyze VEGF activity, and the expression of poly (ADP-ribose) polymerase (PARP), phosphorylated protein kinase B (Akt), Bcl-2-associated X protein (Bax), hypoxia inducible factor (HIF)-1α and inter-cellular adhesion molecule-1 (ICAM-1). Tetrandrine effectively suppressed the growth of and induced apoptosis in A549 lung cancer cells. The expression of PARP, Bax, intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF) was significantly upregulated, and the phosphorylation of Akt and expression of HIF-1α was significantly suppressed in A549 lung cancer cells. Therefore, tetrandrine may suppress cell viability and induce apoptosis via the VEGF/HIF-1α/ICAM-1 signaling pathway. PMID:29849794

Anti-adhesion Property of the Potential Probiotic Strain Lactobacillus fermentum 8711 Against Methicillin-Resistant Staphylococcus aureus (MRSA).

PubMed

Jayashree, Sathyanarayanan; Karthikeyan, Raman; Nithyalakshmi, Sampath; Ranjani, Jothi; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2018-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen and one of the leading causes of nosocomial infection worldwide. Probiotic bacteria play a significant role in preventive or therapeutic interventions of gastrointestinal infections in human as well as animals. In this study, we have investigated the adhesion property of the probiotic strain Lactobacillus fermentum MTCC 8711 and its ability to prevent the adhesion of MRSA to human colon adenocarcinoma cells, Caco-2. We have shown that L. fermentum could efficiently adhere to the Caco-2 cells. Also, we have shown that L. fermentum significantly reduced MRSA adhesion to Caco-2 cells. Three types of experiments were performed to assess the anti-adhesion property of L. fermentum against MRSA. Inhibition (Caco-2 cells were pre-treated with L. fermentum , and subsequently MRSA was added), competition (both L. fermentum and MRSA were added to Caco-2 cells simultaneously), and displacement or exclusion (Caco-2 cells were pre-treated with MRSA, and subsequently L. fermentum was added). In all three experiments, adhesion of MRSA was significantly reduced. Interestingly, L. fermentum could efficiently displace the adhered MRSA, and hence this probiotic can be used for therapeutic applications also. In cytotoxicity assay, we found that L. fermentum per se was not cytotoxic, and also significantly reduced the MRSA-induced cytotoxicity. The protective effect occurred without affecting Caco-2 cell morphology and viability.

Cooperation of HIF- and NCAM-mediated mechanisms in cell viability of hippocampal cultures after oxygen-glucose deprivation.

PubMed

Lushnikova, Iryna; Nikandrova, Yelyzaveta; Skibo, Galyna

2017-10-01

Neurodegenerative diseases of different genesis are the result of cellular damages including those caused by oxygen and glucose deficit. Neuronal survival or death in brain pathologies depends on a variety of interrelated molecular mechanisms. A key role in modulation of neuron viability belongs to HIF (hypoxia-inducible factor) and NCAM (neural cell adhesion molecules) signaling pathways. In this work, we used organotypic and dissociated hippocampal cultures to analyze cell viability and HIF-1α immunopositive (HIF-1α + ) signal after 30 min oxygen-glucose deprivation (OGD) followed by 24 h of reoxygenation in the presence of FGL (synthetic NCAM-derived mimetic peptide). According to LDH- and MTS-assay of cell viability, FGL showed a neuroprotective effect, which was attributed to the association with FGFR. We showed that these effects correlated with changes of the HIF-1α + level suggesting the communications of HIF and NCAM signaling pathways. These data extend our knowledge of neurodegeneration mechanisms and open additional potential for the development of neuroprotection strategies. © 2017 International Federation for Cell Biology.

Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds as a Cell Delivery Vehicle: Characterization of PC12 Cell Response

PubMed Central

Zustiak, Silviya P.; Pubill, Stephanie; Ribeiro, Andreia; Leach, Jennie B.

2013-01-01

The central nervous system (CNS) has a low intrinsic potential for regeneration following injury and disease, yet neural stem/progenitor cell (NPC) transplants show promise to provide a dynamic therapeutic in this complex tissue environment. Moreover, biomaterial scaffolds may improve the success of NPC-based therapeutics by promoting cell viability and guiding cell response. We hypothesized that a hydrogel scaffold could provide a temporary neurogenic environment that supports cell survival during encapsulation, and degrades completely in a temporally controlled manner to allow progression of dynamic cellular processes such as neurite extension. We utilized PC12 cells as a model cell line with an inducible neuronal phenotype to define key properties of hydrolytically-degradable poly(ethylene glycol) hydrogel scaffolds that impact cell viability and differentiation following release from the degraded hydrogel. Adhesive peptide ligands (RGDS, IKVAV or YIGSR), were required to maintain cell viability during encapsulation; as compared to YIGSR, the RGDS and IKVAV ligands were associated with a higher percentage of PC12 cells that differentiated to the neuronal phenotype following release from the hydrogel. Moreover, among the hydrogel properties examined (e.g., ligand type, concentration), total polymer density within the hydrogel had the most prominent effect on cell viability, with densities above 15% w/v leading to decreased cell viability likely due to a higher shear modulus. Thus, by identifying key properties of degradable hydrogels that affect cell viability and differentiation following release from the hydrogel, we lay the foundation for application of this system towards future applications of the scaffold as a neural cell delivery vehicle. PMID:24474590

The effect of nicotine and cotinine on human gingival fibroblasts attachment to root surfaces.

PubMed

Esfahrood, Zeinab Rezaei; Zamanian, Amirhosein; Torshabi, Maryam; Abrishami, Maryam

2015-09-01

Different compounds of smoking (e.g., nicotine and cotinine) are risk factors for various diseases such as oral cancer and periodontal diseases. Some studies reported the negative effects of nicotine on cell proliferation and differentiation. The present in vitro study assessed the effects of nicotine and cotinine (long-acting metabolite of nicotine) on the attachment and viability of human gingival fibroblast (HGF) cells to tooth root surfaces. A total of 70 teeth specimens were placed into 48-well culture plates and covered with HGF cell suspension, in complete Dulbecco's modified Eagle's medium culture medium containing 1 nM, 1 μm, 1 mM, and 5 mM of nicotine and cotinine concentrations. Cellular attachment and viability measured using an MTT assay and a scanning electron microscope were used for cell morphological evaluation. After 24 h, low (nanomolar and micromolar) and high concentrations (millimolar) of nicotine and cotinine caused a significant reduction in the initial cell adhesion in comparison with the control group, but no significant difference was observed between the nicotine and the cotinine groups (p<0.05). Dentally attached cells with low concentrations of nicotine and cotinine proliferated 48 h after exposure, the same as the control group. However, dentally attached cells with high concentrations of nicotine and cotinine (especially 5 mM) did not proliferate 24 h after exposure (p<0.05). Low concentrations of nicotine and cotinine caused a reduction in the initial cell adhesion. However, no significant adverse effects on the proliferation of attached cells were seen in the longer period. High concentrations of nicotine and cotinine have adverse effects on the cell adhesion and proliferation of HGF cells.

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds.

PubMed

Gomes, S R; Rodrigues, G; Martins, G G; Henriques, C M R; Silva, J C

2013-04-01

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solution's properties (surface tension, electrical conductivity and viscosity) and scaffold's properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation. Copyright © 2012 Elsevier B.V. All rights reserved.

CRYOPRESERVATION EFFECTS ON RECOMBINANT MYOBLASTS ENCAPSULATED IN ADHESIVE ALGINATE HYDROGELS

PubMed Central

Ahmad, Hajira F.; Sambanis, Athanassios

2013-01-01

Cell encapsulation in hydrogels is widely used in tissue engineering applications, including encapsulation of islets or other insulin-secreting cells in pancreatic substitutes. Use of adhesive, bio-functionalized hydrogels is receiving increasing attention, as cell-matrix interactions in 3-D can be important for various cell processes. With pancreatic substitutes, studies have indicated benefits of 3-D adhesion on the viability and/or function of insulin-secreting cells. As long-term storage of microencapsulated cells is critical for their clinical translation, cryopreservation of cells in hydrogels is actively being investigated. Previous studies have examined the cryopreservation response of cells encapsulated in non-adhesive hydrogels using conventional freezing and/or vitrification (ice-free cryopreservation), however, none have systematically compared the two cryopreservation methods with cells encapsulated within an adhesive 3-D environment. The latter would be significant, as evidence suggests adhesion influences cellular response to cryopreservation. Thus, the objective of this study was to determine the response to conventional freezing and vitrification of insulin-secreting cells encapsulated in an adhesive biomimetic hydrogel. Recombinant insulin-secreting C2C12 myoblasts were encapsulated in oxidized RGD-alginate and cultured 1 or 4 days post-encapsulation, cryopreserved, and assessed up to 3 days post-warming for metabolic activity and insulin secretion, and one day post-warming for cell morphology. Besides certain transient differences of the vitrified group relative to the Fresh control, both conventional freezing and vitrification maintained metabolism, secretion and morphology of the recombinant C2C12 cells. Thus, due to a simpler procedure and slightly superior results, conventional freezing is recommended over vitrification for the cryopreservation of C2C12 cells in oxidized RGD-modified alginate. PMID:23499987

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

PubMed

Lin, Chunbo; Shen, Maorong; Chen, Weiping; Li, Xiaofeng; Luo, Daoming; Cai, Jinhong; Yang, Yuan

2015-11-01

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40 yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Resveratrol Regulates Colorectal Cancer Cell Invasion by Modulation of Focal Adhesion Molecules

PubMed Central

Buhrmann, Constanze; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi

2017-01-01

Resveratrol, a safe and multi-targeted agent, has been associated with suppression of survival, proliferation and metastasis of cancer, however, the underlying mechanisms for its anti-cancer activity, particularly on cellular signaling during cancer cell migration still remain poorly understood. We investigated the invasion response of two human colorectal cancer (CRC) cells (HCT116 and SW480) to resveratrol and studied the effect of specific pharmacological inhibitors, cytochalasin D (CytD) and focal adhesion kinase-inhibitor (FAK-I) on FAK, cell viability and migration in CRC. We found that resveratrol altered cell phenotype of both CRC cells, reduced cell viability and the results were comparable to FAK-I and CytD. These effects of resveratrol were associated with marked Sirt1 up-regulation, FAK down-regulation, inhibition of focal adhesion and potentiation of effects by combinatorial treatment of resveratrol and inhibitors. Interestingly, inhibition of FAK with FAK-I or treatment with CytD suppressed resveratrol-induced Sirt1 up-regulation and markedly down-regulated FAK expression. Resveratrol or combination treatment with inhibitors significantly activated caspase-3 and potentiated apoptosis. Moreover, resveratrol suppressed invasion and colony forming capacity, cell proliferation, β1-Integrin expression and activation of FAK of cells in alginate tumor microenvironment, similar to FAK-I or CytD. Finally, we demonstrated that resveratrol, FAK-I or CytD inhibited activation of NF-κB, suppressed NF-κB-dependent gene end-products involved in invasion, metastasis, and apoptosis; and these effects of resveratrol were potentiated by combination treatment with FAK-I or CytD. Our data illustrated that the anti-invasion effect of resveratrol by inhibition of FAK activity has a potential beneficial role in disease prevention and therapeutic management of CRC. PMID:28953264

Resveratrol Regulates Colorectal Cancer Cell Invasion by Modulation of Focal Adhesion Molecules.

PubMed

Buhrmann, Constanze; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi

2017-09-27

Resveratrol, a safe and multi-targeted agent, has been associated with suppression of survival, proliferation and metastasis of cancer, however, the underlying mechanisms for its anti-cancer activity, particularly on cellular signaling during cancer cell migration still remain poorly understood. We investigated the invasion response of two human colorectal cancer (CRC) cells (HCT116 and SW480) to resveratrol and studied the effect of specific pharmacological inhibitors, cytochalasin D (CytD) and focal adhesion kinase-inhibitor (FAK-I) on FAK, cell viability and migration in CRC. We found that resveratrol altered cell phenotype of both CRC cells, reduced cell viability and the results were comparable to FAK-I and CytD. These effects of resveratrol were associated with marked Sirt1 up-regulation, FAK down-regulation, inhibition of focal adhesion and potentiation of effects by combinatorial treatment of resveratrol and inhibitors. Interestingly, inhibition of FAK with FAK-I or treatment with CytD suppressed resveratrol-induced Sirt1 up-regulation and markedly down-regulated FAK expression. Resveratrol or combination treatment with inhibitors significantly activated caspase-3 and potentiated apoptosis. Moreover, resveratrol suppressed invasion and colony forming capacity, cell proliferation, β1-Integrin expression and activation of FAK of cells in alginate tumor microenvironment, similar to FAK-I or CytD. Finally, we demonstrated that resveratrol, FAK-I or CytD inhibited activation of NF-κB, suppressed NF-κB-dependent gene end-products involved in invasion, metastasis, and apoptosis; and these effects of resveratrol were potentiated by combination treatment with FAK-I or CytD. Our data illustrated that the anti-invasion effect of resveratrol by inhibition of FAK activity has a potential beneficial role in disease prevention and therapeutic management of CRC.

Porcine circovirus 2 (PCV2) increases the expression of endothelial adhesion/junction molecules.

PubMed

Marks, Fernanda S; Almeida, Laura L; Driemeier, David; Canal, Cláudio; Barcellos, David E S N; Guimarães, Jorge A; Reck, José

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus disease, a complex multisystem syndrome in domestic pigs. Despite the significant economic losses caused by porcine circovirus disease, the mechanisms of pathogenesis underlying the clinical findings remain largely unclear. As various reports have highlighted the potential key role of vascular lesions in the pathogenesis of porcine circovirus disease, the aim of this work was to investigate effects of PCV2 infection on vascular endothelial cells, focusing on cell viability and expression of adhesion/junction molecules. PCV2 infection reduced endothelial cell viability, while viral infection did not affected the viability of several other classical cell lines. Also, PCV2 infection in endothelial cells displayed a dual/biphasic effect: initially, infection increased ICAM-1 expression, which can favor leukocyte recruitment and emigration to tissues and possibly inducing characteristic porcine circovirus disease inflammatory lesions; then, secondarily, infection caused an increase in zonula occludens 1 tight junction protein (ZO-1) expression, which in turn can result in difficulties for cell traffic across the endothelium and a potential impairment the immune response in peripheral tissues. These virus-induced endothelial changes could directly impact the inflammatory process of porcine circovirus disease and associated vascular/immune system disturbances. Data suggest that, among the wide range of effects induced by PCV2 on the host, endothelial modulation can be a pivotal process which can help to explain PCV2 pathogenesis in some porcine circovirus disease presentations. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells.

PubMed

Hebling, J; Bianchi, L; Basso, F G; Scheffel, D L; Soares, D G; Carrilho, M R O; Pashley, D H; Tjäderhane, L; de Souza Costa, C A

2015-04-01

To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm(2)) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney's tests (p < 0.05). Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Fabrication and evaluation of novel zeolite membranes to control the neoplastic activity and anti-tumoral drug treatments in human breast cancer cells. Part 1: Synthesis and characterization of Pure Zeolite Membranes and Mixed Matrix Membranes for adhesion and growth of cancer cells.

PubMed

Tavolaro, Palmira; Martino, Guglielmo; Andò, Sebastiano; Tavolaro, Adalgisa

2016-12-01

Novel pure and hybrid zeolite membranes were prepared with appropriate different physicochemical characteristics such as frameworks, hydrophilicity, crystal size, chemical composition, acid-base properties (Point of Zero Charge, PZC) and surface morphology and used in inorganic cell/scaffold constructs. Because the control of cell interactions, as the adhesion, proliferation, remodelling and mobility, is important for differentiation and progression of tumors, this work focused on response of cancer cells adhered and grown on synthesized zeolite surfaces in order to study the influence of these scaffolds in controlled conditions. We have selected the MCF-7 and MDA-MB-231 human breast cancer cell line as model tumor cell lines. This study showed that all the zeolite membranes synthesized are excellent scaffolds because they are very selective materials to support the adhesion and growth of neoplastic cells. All zeolite scaffolds were characterized by FESEM, FTIR ATR, XRD, AFM, PZC and contact angle analyses. Cell adhesion, viability and morphology were measured by count, MTT assay and FESEM microphotography analysis, at various incubation times. Copyright © 2016. Published by Elsevier B.V.

Regulation of RAW 264.7 macrophages behavior on anodic TiO2 nanotubular arrays

NASA Astrophysics Data System (ADS)

Yao, Shenglian; Feng, Xujia; Li, Wenhao; Wang, Lu-Ning; Wang, Xiumei

2017-12-01

Titanium (Ti) implants with TiO2 nanotubular arrays on the surface could regulate cells adhesion, proliferation and differentiation to determine the bone integration. Additionally, the regulation of immune cells could improve osteogenesis or lead in appropriate immune reaction. Thus, we evaluate the behavior of RAW264.7 macrophages on TiO2 nanotubular arrays with a wide range diameter (from 20 to 120 nm) fabricated by an electrochemical anodization process. In this work, the proliferation, cell viability and cytokine/chemokine secretion were evaluated by CCK-8, live/dead staining and ELISA, respectively. SEM and confocal microscopy were used to observe the adhesion morphology. Results showed that the small size nanotube surface was benefit for the macrophages adhesion and proliferation, while larger size surface could reduce the inflammatory response. These findings contribute to the design of immune-regulating Ti implants surface that supports successful implantation.

Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin, sodium fluoride, melanocyte-stimulating hormone.

PubMed

Zijah, Vahid; Salehi, Roya; Aghazadeh, Marziyeh; Samiei, Mohammad; Alizadeh, Effat; Davaran, Soodabeh

2017-06-01

Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.

Synthesis and characterization of nanostructured CaSiO3 biomaterial

NASA Astrophysics Data System (ADS)

Jagadale, Pramod N.; Kulal, Shivaji R.; Joshi, Meghanath G.; Jagtap, Pramod P.; Khetre, Sanjay M.; Bamane, Sambhaji R.

2013-04-01

Here we report a successful preparation of nanostructured calcium silicate by wet chemical approach. The synthesized sample was characterized by various physico-chemical methods. Thermal stability was investigated using thermo-gravimetric and differential thermal analysis (TG-DTA). Structural characterization of the sample was carried out by the X-ray diffraction technique (XRD) which confirmed its single phase hexagonal structure. Transmission electron microscopy (TEM) was used to study the nanostructure of the ceramics while homogeneous grain distribution was revealed by scanning electron microscopy studies (SEM). The elemental analysis data obtained from energy dispersive X-ray spectroscopy (EDAX) were in close agreement with the starting composition used for the synthesis. Superhydrophilic nature of CaSiO3 was investigated at room temperature by sessile drop technique. Effect of porous nanosized CaSiO3 on early adhesion and proliferation of human bone marrow mesenchymal stem cells (BMMSCs) and cord blood mesenchymal stem (CBMSCs) cells was measured in vitro. MTT cytotoxicity test and cell adhesion test showed that the material had good biocompatibility and promoted cell viability and cell proliferation. It has been stated that the cell viability and proliferation are significantly affected by time and concentration of CaSiO3. These findings indicate that the CaSiO3 ceramics has good biocompatibility and that it is promising as a biomaterial.

Storage effect on viability and biofunctionality of human adipose tissue-derived stromal cells.

PubMed

Falah, Mizied; Rayan, Anwar; Srouji, Samer

2015-09-01

In our recent studies, the transplantation of human adipose tissue-derived stromal cells (ASCs) has shown promise for treatment of diseases related to bone and joint disorders. For the current clinical applications, ASCs were formulated and suspended in PlasmaLyte A supplemented with heparin, glucose and human serum albumin, balanced to pH 7.4 with sodium bicarbonate. This cell solution constitutes 20% of the overall transplanted mixture and is supplemented with hyaluronic acid (60%) and OraGraft particles (20%). We intended to investigate the effect of this transplantation mixture on the viability and biofunctionality of ASCs in bone formation. Freshly harvested cells were resuspended and incubated in the indicated mixture for up to 48 h at 4°C. Cell viability was assessed using trypan blue and AlamarBlue, and cell functionality was determined by quantifying their adhesion rate in vitro and bone formation in an ectopic mouse model. More than 80% of the ASCs stored in the transplantation mixture were viable for up to 24 h. Cell viability beyond 24 h in storage decreased to approximately 50%. In addition, an equal degree of bone formation was observed between the cells transplanted following incubation in transplantation mixture for up to 24 h and zero-time non-incubated cells (control). The viability and functionality of ASCs stored in the presented formulation will make such cell therapy accessible to larger and more remote populations. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Multiscale Morphology of Organic Semiconductor Thin Films Controls the Adhesion and Viability of Human Neural Cells

PubMed Central

Tonazzini, I.; Bystrenova, E.; Chelli, B.; Greco, P.; Stoliar, P.; Calò, A.; Lazar, A.; Borgatti, F.; D'Angelo, P.; Martini, C.; Biscarini, F.

2010-01-01

Abstract We investigate how multiscale morphology of functional thin films affects the in vitro behavior of human neural astrocytoma 1321N1 cells. Pentacene thin film morphology is precisely controlled by means of the film thickness, Θ (here expressed in monolayers (ML)). Fluorescence and atomic force microscopy allow us to correlate the shape, adhesion, and proliferation of cells to the morphological properties of pentacene films controlled by saturated roughness, σ, correlation length, ξ, and fractal dimension, df. At early incubation times, cell adhesion exhibits a transition from higher to lower values at Θ ≈ 10 ML. This is explained using a model of conformal adhesion of the cell membrane onto the growing pentacene islands. From the model fitting of the data, we show that the cell explores the surface with a deformation of the membrane whose minimum curvature radius is 90 (± 45) nm. The transition in the adhesion at ∼10 ML arises from the saturation of ξ accompanied by the monotonic increase of σ, which leads to a progressive decrease of the pentacene local radius of curvature and hence to the surface area accessible to the cell. Cell proliferation is also enhanced for Θ < 10 ML, and the optimum morphology parameter ranges for cell deployment and growth are σ ≤ 6 nm, ξ > 500 nm, and df > 2.45. The characteristic time of cell proliferation is τ ≈ 10 ± 2 h. PMID:20550892

Ganoderma lucidum (Reishi) inhibits cancer cell growth and expression of key molecules in inflammatory breast cancer.

PubMed

Martínez-Montemayor, Michelle M; Acevedo, Raysa Rosario; Otero-Franqui, Elisa; Cubano, Luis A; Dharmawardhane, Suranganie F

2011-01-01

Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell-cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic.

A novel and selective inhibitor of PKC ζ potently inhibits human breast cancer metastasis in vitro and in mice.

PubMed

Wu, Jing; Liu, Shuye; Fan, Zhijuan; Zhang, Lei; Tian, Yaqiong; Yang, Rui

2016-06-01

Cell motility and chemotaxis play pivotal roles in the process of tumor development and metastasis. Protein kinase C ζ (PKC ζ) mediates epidermal growth factor (EGF)-stimulated chemotactic signaling pathway through regulating cytoskeleton rearrangement and cell adhesion. The purpose of this study was to develop anti-PKC ζ therapeutics for breast cancer metastasis. In this study, a novel and high-efficient PKC ζ inhibitor named PKCZI195.17 was screened out through a substrate-specific strategy. MTT assay was used to determine the cell viability of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells while under PKCZI195.17 treatment. Wound-healing, chemotaxis, and Matrigel invasion assays were performed to detect the effects of PKCZI195.17 on breast cancer cells migration and invasion. Adhesion, actin polymerization, and Western blotting were performed to detect the effects of PKCZI195.17 on cells adhesion and actin polymerization, and explore the downsteam signaling mechanisms involved in PKC ζ inhibition. MDA-MB-231 xenograft was used to measure the in vivo anti-metastasis efficacy of PKCZI195.17. The compound PKCZI195.17 selectively inhibited PKC ζ kinase activity since it failed to inhibit PKC α, PKC β, PKC δ, PKC η, AKT2, as well as FGFR2 activity. PKCZI195.17 significantly impaired spontaneous migration, chemotaxis, and invasion of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells, while PKCZI195.17 did not obviously inhibited cells viability. PKCZI195.17 also inhibited cells adhesion and actin polymerization through attenuating the phosphorylations of integrin β1, LIMK, and cofilin, which might be the downstream effectors of PKC ζ-mediated chemotaxis in MDA-MB-231 cells. Furthermore, PKCZI195.17 suppressed the breast cancer metastasis and increased the survival time of breast tumor-bearing mice. In summary, PKCZI195.17 was a PKC ζ-specific inhibitor which dampened cancer cell migration and metastasis and may serve as a novel therapeutic drug for breast cancer metastasis.

Role of nanostructured gold surfaces on monocyte activation and Staphylococcus epidermidis biofilm formation

PubMed Central

Svensson, Sara; Forsberg, Magnus; Hulander, Mats; Vazirisani, Forugh; Palmquist, Anders; Lausmaa, Jukka; Thomsen, Peter; Trobos, Margarita

2014-01-01

The role of material surface properties in the direct interaction with bacteria and the indirect route via host defense cells is not fully understood. Recently, it was suggested that nanostructured implant surfaces possess antimicrobial properties. In the current study, the adhesion and biofilm formation of Staphylococcus epidermidis and human monocyte adhesion and activation were studied separately and in coculture in different in vitro models using smooth gold and well-defined nanostructured gold surfaces. Two polystyrene surfaces were used as controls in the monocyte experiments. Fluorescent viability staining demonstrated a reduction in the viability of S. epidermidis close to the nanostructured gold surface, whereas the smooth gold correlated with more live biofilm. The results were supported by scanning electron microscopy observations, showing higher biofilm tower formations and more mature biofilms on smooth gold compared with nanostructured gold. Unstimulated monocytes on the different substrates demonstrated low activation, reduced gene expression of pro- and anti-inflammatory cytokines, and low cytokine secretion. In contrast, stimulation with opsonized zymosan or opsonized live S. epidermidis for 1 hour significantly increased the production of reactive oxygen species, the gene expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-10, as well as the secretion of TNF-α, demonstrating the ability of the cells to elicit a response and actively phagocytose prey. In addition, cells cultured on the smooth gold and the nanostructured gold displayed a different adhesion pattern and a more rapid oxidative burst than those cultured on polystyrene upon stimulation. We conclude that S. epidermidis decreased its viability initially when adhering to nanostructured surfaces compared with smooth gold surfaces, especially in the bacterial cell layers closest to the surface. In contrast, material surface properties neither strongly promoted nor attenuated the activity of monocytes when exposed to zymosan particles or S. epidermidis. PMID:24550671

Ganoderma lucidum (Reishi) Inhibits Cancer Cell Growth and Expression of Key Molecules in Inflammatory Breast Cancer

PubMed Central

Martínez-Montemayor, Michelle M.; Acevedo, Raysa Rosario; Otero-Franqui, Elisa; Cubano, Luis. A.; Dharmawardhane, Suranganie F.

2011-01-01

Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell–cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic. PMID:21888505

The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions.

PubMed

Sepulveda-Medina, Paola; Katsenovich, Yelena; Musaramthota, Vishal; Lee, Michelle; Lee, Brady; Dua, Rupak; Lagos, Leonel

2015-06-01

Past disposal practices at nuclear production facilities have led to the release of liquid waste into the environment creating multiple radionuclide plumes. Microorganisms are known for the ability to interact with radionuclides and impact their mobility in soils and sediments. Gram-positive Arthrobacter sp. are one of the most common bacterial groups in soils and are found in large numbers in subsurface environments contaminated with radionuclides. This study experimentally analyzed changes on the bacteria surface at the nanoscale level after uranium exposure and evaluated the effect of aqueous bicarbonate ions on U(VI) toxicity of a low uranium-tolerant Arthrobacter oxydans strain G968 by investigating changes in adhesion forces and cell dimensions via atomic force microscopy (AFM). Experiments were extended to assess cell viability by the Live/Dead BacLight Bacterial Viability Kit (Molecular Probes) and quantitatively illustrate the effect of uranium exposure in the presence of varying concentrations of bicarbonate ions. AFM and viability studies showed that samples containing bicarbonate were able to withstand uranium toxicity and remained viable. Samples containing no bicarbonate exhibited deformed surfaces and a low height profile, which, in conjunction with viability studies, indicated that the cells were not viable. Copyright © 2015 Institut Pasteur. All rights reserved.

CD151-mediated adhesion is crucial to osteosarcoma pulmonary metastasis

PubMed Central

Sun, Mengxiong; Zhou, Chenghao; Chen, Jian; Yin, Fei; Wang, Hongsheng; Lin, Binhui; Zuo, Dongqing; Li, Suoyuan; Feng, Lijin; Duan, Zhenfeng; Cai, Zhengdong; Hua, Yingqi

2016-01-01

CD151, a tetraspanin family protein involved in cell-cell and cell-extracellular matrix interaction, is differentially expressed in osteosarcoma cell membranes. Thus, this study aimed to investigate the role of CD151 in osteosarcoma metastasis. We analyzed CD151 expression in patient tissue samples using immunohistochemistry. CD151 expression was also silenced with shRNA in osteosarcoma cells of high metastatic potential, and cell adhesion, migration and invasion were evaluated in vitro and pulmonary metastasis was investigated in vivo. Mediators of cell signaling pathways were also examined following suppression of CD151 expression. Overall survival for patients with low versus high CD151 expression level was 94 vs. 41 months (p=0.0451). CD151 expression in osteosarcoma cells with high metastatic potential was significantly higher than in those with low metastatic potential (p<0.001). shRNA-mediated silencing of CD151 did not influence cell viability or proliferation; however, cell adhesion, migration and invasion were all inhibited (all p<0.001). In mice inoculated with shRNA-transduced osteosarcoma cells, the number and size of lung metastatic lesions were reduced compared to the mice inoculated with control-shRNA transduced cells (p<0.001). In addition, CD151 knockdown significantly reduced Akt, p38, and p65 phosphorylation as well as focal adhesion kinase, integrin β1, p70s6, and p-mTOR levels. Taken together, CD151 induced osteosarcoma metastasis likely by regulating cell function through adhesion signaling. Further studies are necessary to fully explore the diagnostic and prognostic value of determining CD151 expression in osteosarcoma patients. PMID:27556355

Biocompatibility and bond degradation of poly-acrylic acid coated copper iodide-adhesives.

PubMed

ALGhanem, Adi; Fernandes, Gabriela; Visser, Michelle; Dziak, Rosemary; Renné, Walter G; Sabatini, Camila

2017-09-01

To investigate the effect of poly-acrylic acid (PAA) copper iodide (CuI) adhesives on bond degradation, tensile strength, and biocompatibility. PAA-CuI particles were incorporated into Optibond XTR, Optibond Solo and XP Bond in 0.1 and 0.5mg/ml. Clearfil SE Protect, an MDPB-containing adhesive, was used as control. The adhesives were applied to human dentin, polymerized and restored with composite in 2mm-increments. Resin-dentin beams (0.9±0.1mm 2 ) were evaluated for micro-tensile bond strength after 24h, 6 months and 1year. Hourglass specimens (10×2×1mm) were evaluated for ultimate tensile strength (UTS). Cell metabolic function of human gingival fibroblast cells exposed to adhesive discs (8×1mm) was assessed with MTT assay. Copper release from adhesive discs (5×1mm) was evaluated with UV-vis spectrophotometer after immersion in 0.9% NaCl for 1, 3, 5, 7, 10, 14, 21 and 30 days. SEM, EDX and XRF were conducted for microstructure characterization. XTR and Solo did not show degradation when modified with PAA-CuI regardless of the concentration. The UTS for adhesives containing PAA-CuI remained unaltered relative to the controls. The percent viable cells were reduced for Solo 0.5mg/ml and XP 0.1 or 0.5mg/ml PAA-CuI. XP demonstrated the highest ion release. For all groups, the highest release was observed at days 1 and 14. PAA-CuI particles prevented the bond degradation of XTR and Solo after 1year without an effect on the UTS for any adhesive. Cell viability was affected for some adhesives. A similar pattern of copper release was demonstrated for all adhesives. Copyright © 2017. Published by Elsevier Ltd.

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

PubMed

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

2018-04-30

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

Optimized delivery of skin keratinocytes by aerosolization and suspension in fibrin tissue adhesive.

PubMed

Harkin, Damien G; Dawson, Rebecca A; Upton, Zee

2006-01-01

Aerosolized suspensions of keratinocytes provide a potential therapy for wounds, but the effects of aerosolization on cell viability remain unclear. Likewise, little is known of the resulting cell distribution pattern and how this compares to the density required for epithelialization. The potential benefits of cospraying cells in the presence of fibrin adhesive are equally uncertain. Thus, in the present study we have optimized conditions for the aerosolization of cultured keratinocytes using a device (Tissomat) that supports the option for coapplication with fibrin (Tisseel). Cell viability was unaffected when sprayed at 10 psi, but a significant reduction in metabolic activity, as determined by the methylthiazoyldiphenol-tetrazolium assay, was observed at higher pressure. Bursts of 0.2 mL cell suspension (1.5x10(6)/mL) delivered from a height of 10 cm was sufficient to epithelialize an area of 10-15 cm2 within 7 days in vitro. Confluent areas corresponded to those with a density of 5,000-10,000 cells/cm2 at 24 hours. Optimal cell growth in Tisseel was achieved through dilution of fibrinogen (1-3 mg/mL) and thrombin (2-5 IU/mL). This optimized formulation eliminated fluid run-off postspraying and stimulated a twofold increase in cellular response. Therefore, our in vitro data supports the theory that aerosolized suspensions of keratinocytes in fibrin will benefit healing.

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow.

PubMed

van der Mei, Henny C; Atema-Smit, Jelly; Jager, Debbie; Langworthy, Don E; Collias, Dimitris I; Mitchell, Michael D; Busscher, Henk J

2008-07-01

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination. (c) 2008 Wiley Periodicals, Inc.

A method to screen and evaluate tissue adhesives for joint repair applications

PubMed Central

2012-01-01

Background Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. Method Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. Results Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). Conclusion The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings. PMID:22984926

xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

PubMed

Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan

2014-01-01

The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

Thrombomodulin exerts cytoprotective effect on low-dose UVB-irradiated HaCaT cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwata, Masahiro; Laboratory of Vascular Medicine, Department of Cardiovascular and Respiratory Disorders Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520; Kawahara, Ko-ichi

Thrombomodulin (TM) is an endothelial cell surface anticoagulant glycoprotein that performs antimetastatic, angiogenic, adhesive, and anti-inflammatory functions in various tissues. It is also expressed in epidermal keratinocytes. We found that a physiological dose (10 mJ/cm{sup 2}) of mid-wavelength ultraviolet irradiation (UVB) significantly induced TM expression via the p38mitogen-activated protein kinase (MAPK)/cyclic AMP response element (CRE) signaling pathway in the epidermal keratinocyte cell line HaCaT; this shows that TM regulates the survival of HaCaT cells. SB203580, a p38MAPK inhibitor, significantly decreased TM expression and the viability of cells exposed to UVB. Furthermore, overexpression of TM markedly increased cell viability, and itmore » was abrogated by TM small interfering RNA (siRNA), suggesting that TM may play an important role in exerting cytoprotective effect on epidermal keratinocytes against low-dose UVB.« less

Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

PubMed Central

Schleicher, Martina; Hansmann, Jan; Elkin, Bentsian; Kluger, Petra J.; Liebscher, Simone; Huber, Agnes J. T.; Fritze, Olaf; Schille, Christine; Müller, Michaela; Schenke-Layland, Katja; Seifert, Martina; Walles, Heike; Wendel, Hans-Peter; Stock, Ulrich A.

2012-01-01

In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene)) coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2) and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P < 0.05). Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization. PMID:22481939

nDEP-driven cell patterning and bottom-up construction of cell aggregates using a new bioelectronic chip.

PubMed

Menad, S; Franqueville, L; Haddour, N; Buret, F; Frenea-Robin, M

2015-04-01

Creating cell aggregates of controlled size and shape and patterning cells on substrates using a bottom-up approach constitutes important challenges for tissue-engineering applications and studies of cell-cell interactions. In this paper, we report nDEP (negative dielectrophoresis) driven assembly of cells as compact aggregates or onto defined areas using a new bioelectronic chip. This chip is composed of a quadripolar electrode array obtained using coplanar electrodes partially covered with a thin, micropatterned PDMS membrane. This thin PDMS layer was coated with poly-L-lysine and played the role of adhesive substrate for cell patterning. For the formation of detachable cell aggregates, the PDMS was not pretreated and cells were simply immobilized into assemblies maintained by cell-cell adhesion after the electric field removal. Cell viability after exposition to DEP buffer was also assessed, as well as cell spreading activity following DEP-driven assembly. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing

PubMed Central

Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

2017-01-01

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%–3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process. PMID:28772551

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing.

PubMed

Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

2017-02-16

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%-3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

PubMed Central

Gabriele, Morena; Del Prato, Stefano; Pucci, Laura

2017-01-01

Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS) orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs), bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG), on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER) stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation. PMID:28386305

Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawal, Akeem O.; Zhang, Min; Dittmar, Michael

Diesel exhaust particles (DEPs) are a major component of diesel emissions, responsible for a large portion of their toxicity. In this study, we examined the toxic effects of DEPs on endothelial cells and the role of DEP-induced heme oxygenase-1 (HO-1) expression. Human microvascular endothelial cells (HMECs) were treated with an organic extract of DEPs from an automobile engine (A-DEP) or a forklift engine (F-DEP) for 1 and 4 h. ROS generation, cell viability, lactate dehydrogenase leakage, expression of HO-1, inflammatory genes, cell adhesion molecules and unfolded protein respone (UPR) gene were assessed. HO-1 expression and/or activity were inhibited by siRNAmore » or tin protoporphyrin (Sn PPIX) and enhanced by an expression plasmid or cobalt protoporphyrin (CoPPIX). Exposure to 25 μg/ml of A-DEP and F-DEP significantly induced ROS production, cellular toxicity and greater levels of inflammatory and cellular adhesion molecules but to a different degree. Inhibition of HO-1 enzymatic activity with SnPPIX and silencing of the HO-1 gene by siRNA enhanced DEP-induced ROS production, further decreased cell viability and increased expression of inflammatory and cell adhesion molecules. On the other hand, overexpression of the HO-1 gene by a pcDNA 3.1D/V5-HO-1 plasmid significantly mitigated ROS production, increased cell survival and decreased the expression of inflammatory genes. HO-1 expression protected HMECs from DEP-induced prooxidative and proinflammatory effects. Modulation of HO-1 expression could potentially serve as a therapeutic target in an attempt to inhibit the cardiovascular effects of ambient PM. - Highlights: • We examined the role of HO-1 expression on diesel exhaust particle (DEP) in endothelial cells. • DEPs exert cytotoxic and inflammatory effects on human microvascular endothelial cells (HMECs). • DEPs induce HO-1 expression in HMECs. • HO-1 protects against the oxidative stress induced by DEps. • HO-1 attenuates the proinflammatory effects induced by DEPs.« less

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

PubMed Central

Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

2013-01-01

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy. PMID:23626771

Effect of tributyltin on mammalian endothelial cell integrity.

PubMed

Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

2015-01-01

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

PubMed

Carvalho, Eunice B; Maga, Elizabeth A; Quetz, Josiane S; Lima, Ila F N; Magalhães, Hemerson Y F; Rodrigues, Felipe A R; Silva, Antônio V A; Prata, Mara M G; Cavalcante, Paloma A; Havt, Alexandre; Bertolini, Marcelo; Bertolini, Luciana R; Lima, Aldo A M

2012-08-11

Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ko, Jae Hyung; Kim, Yang Hee; Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul

Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3more » dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.« less

Silver deposition on titanium surface by electrochemical anodizing process reduces bacterial adhesion of Streptococcus sanguinis and Lactobacillus salivarius.

PubMed

Godoy-Gallardo, Maria; Rodríguez-Hernández, Ana G; Delgado, Luis M; Manero, José M; Javier Gil, F; Rodríguez, Daniel

2015-10-01

The aim of this study was to determine the antibacterial properties of silver-doped titanium surfaces prepared with a novel electrochemical anodizing process. Titanium samples were anodized with a pulsed process in a solution of silver nitrate and sodium thiosulphate at room temperature with stirring. Samples were processed with different electrolyte concentrations and treatment cycles to improve silver deposition. Physicochemical properties were determined by X-ray photoelectron spectroscopy, contact angle measurements, white-light interferometry, and scanning electron microscopy. Cellular cytotoxicity in human fibroblasts was studied with lactate dehydrogenase assays. The in vitro effect of treated surfaces on two oral bacteria strains (Streptococcus sanguinis and Lactobacillus salivarius) was studied with viable bacterial adhesion measurements and growth curve assays. Nonparametric statistical Kruskal-Wallis and Mann-Whitney U-tests were used for multiple and paired comparisons, respectively. Post hoc Spearman's correlation tests were calculated to check the dependence between bacteria adhesion and surface properties. X-ray photoelectron spectroscopy results confirmed the presence of silver on treated samples and showed that treatments with higher silver nitrate concentration and more cycles increased the silver deposition on titanium surface. No negative effects in fibroblast cell viability were detected and a significant reduction on bacterial adhesion in vitro was achieved in silver-treated samples compared with control titanium. Silver deposition on titanium with a novel electrochemical anodizing process produced surfaces with significant antibacterial properties in vitro without negative effects on cell viability. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bio-inspired enhancement of friction and adhesion at the polydimethylsiloxane-intestine interface and biocompatibility characterization.

PubMed

Zhang, Hongyu; Wang, Yi; Vasilescu, Steven; Gu, Zhibin; Sun, Tao

2017-05-01

An active navigation of self-propelled miniaturized robot along the intestinal tract without injuring the soft tissue remains a challenge as yet. Particularly in this case an effective control of the interfacial friction and adhesion between the material used and the soft tissue is crucial. In the present study, we investigated the frictional and adhesive properties between polydimethylsiloxane (PDMS, microscopically patterned with micro-pillar arrays and non-patterned with a flat surface) and rabbit small intestinal tract using a universal material tester. The friction coefficient-time plot and adhesive force-time plot were recorded during the friction test (sliding speed: 0.25mm/s; normal loading: 0.4N) and adhesion test (preloading: 0.5N; hoisting speed: 2.5×10 -3 mm/s). In addition, biocompatibility of the PDMS samples was characterized in terms of cell morphology (scanning electron microscope) and cell cytotoxicity (alamarBlue assay) using human vascular endothelial cells (HUVECs). The results demonstrated that the interfacial friction (0.27 vs 0.19) and adhesion (34.9mN vs 26.7mN) were greatly increased using microscopically patterned PDMS, in comparison with non-patterned PDMS. HUVECs adhered to and proliferated on non-patterned/microscopically patterned PDMS very well, with a relative cell viability of about 90% following seeding at 1d, 3d, and 5d. The favorable enhancement of the frictional and adhesive properties, along with the excellent biocompatibility of the microscopically patterned PDMS, makes it a propitious choice for clinical application of self-propelled miniaturized robots. Copyright © 2016 Elsevier B.V. All rights reserved.

Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application.

PubMed

Zhang, Fengli; Ren, Huaijuan; Shao, Xiaohu; Zhuang, Chao; Chen, Yantian; Qi, Nianmin

2017-01-01

Adipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2∼8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation. Preservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity. A total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 10 6 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly. In this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 10 6 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy.

Comparison of candidate materials for a synthetic osteo-odonto keratoprosthesis device.

PubMed

Tan, Xiao Wei; Perera, A Promoda P; Tan, Anna; Tan, Donald; Khor, K A; Beuerman, Roger W; Mehta, Jodhbir S

2011-01-05

Osteo-odonto keratoprosthesis is one of the most successful forms of keratoprosthesis surgery for end-stage corneal and ocular surface disease. There is a lack of detailed comparison studies on the biocompatibilities of different materials used in keratoprosthesis. The aim of this investigation was to compare synthetic bioinert materials used for keratoprosthesis surgery with hydroxyapatite (HA) as a reference. Test materials were sintered titanium oxide (TiO(2)), aluminum oxide (Al(2)O(3)), and yttria-stabilized zirconia (YSZ) with density >95%. Bacterial adhesion on the substrates was evaluated using scanning electron microscopy and the spread plate method. Surface properties of the implant discs were scanned using optical microscopy. Human keratocyte attachment and proliferation rates were assessed by cell counting and MTT assay at different time points. Morphologic analysis and immunoblotting were used to evaluate focal adhesion formation, whereas cell adhesion force was measured with a multimode atomic force microscope. The authors found that bacterial adhesion on the TiO(2), Al(2)O(3), and YSZ surfaces were lower than that on HA substrates. TiO(2) significantly promoted keratocyte proliferation and viability compared with HA, Al(2)O(3,) and YSZ. Immunofluorescent imaging analyses, immunoblotting, and atomic force microscope measurement revealed that TiO(2) surfaces enhanced cell spreading and cell adhesion compared with HA and Al(2)O(3). TiO(2) is the most suitable replacement candidate for use as skirt material because it enhanced cell functions and reduced bacterial adhesion. This would, in turn, enhance tissue integration and reduce device failure rates during keratoprosthesis surgery.

Reducing the cytotoxicity of inhalable engineered nanoparticles via in situ passivation with biocompatible materials.

PubMed

Byeon, Jeong Hoon; Park, Jae Hong; Peters, Thomas M; Roberts, Jeffrey T

2015-07-15

The cytotoxicity of model welding nanoparticles was modulated through in situ passivation with soluble biocompatible materials. A passivation process consisting of a spark discharge particle generator coupled to a collison atomizer as a co-flow or counter-flow configuration was used to incorporate the model nanoparticles with chitosan. The tested model welding nanoparticles are inhaled and that A549 cells are a human lung epithelial cell line. Measurements of in vitro cytotoxicity in A549 cells revealed that the passivated nanoparticles had a lower cytotoxicity (>65% in average cell viability, counter-flow) than the untreated model nanoparticles. Moreover, the co-flow incorporation between the nanoparticles and chitosan induced passivation of the nanoparticles, and the average cell viability increased by >80% compared to the model welding nanoparticles. As a more convenient way (additional chitosan generation and incorporation devices may not be required), other passivation strategies through a modification of the welding rod with chitosan adhesive and graphite paste did also enhance average cell viability (>58%). The approach outlined in this work is potentially generalizable as a new platform, using only biocompatible materials in situ, to treat nanoparticles before they are inhaled. Copyright © 2015 Elsevier B.V. All rights reserved.

Bioinspired Pollen-Like Hierarchical Surface for Efficient Recognition of Target Cancer Cells.

PubMed

Wang, Wenshuo; Yang, Gao; Cui, Haijun; Meng, Jingxin; Wang, Shutao; Jiang, Lei

2017-08-01

The efficient recognition and isolation of rare cancer cells holds great promise for cancer diagnosis and prognosis. In nature, pollens exploit spiky structures to realize recognition and adhesion to stigma. Herein, a bioinspired pollen-like hierarchical surface is developed by replicating the assembly of pollen grains, and efficient and specific recognition to target cancer cells is achieved. The pollen-like surface is fabricated by combining filtering-assisted assembly and soft lithography-based replication of pollen grains of wild chrysanthemum. After modification with a capture agent specific to cancer cells, the pollen-like surface enables the capture of target cancer cells with high efficiency and specificity. In addition, the pollen-like surface not only assures high viability of captured cells but also performs well in cell mixture system and at low cell density. This study represents a good example of constructing cell recognition biointerfaces inspired by pollen-stigma adhesion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phloretin attenuates hyperuricemia-induced endothelial dysfunction through co-inhibiting inflammation and GLUT9-mediated uric acid uptake.

PubMed

Liu, Shuyun; Yuan, Yujia; Zhou, Yijie; Zhao, Meng; Chen, Younan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

2017-10-01

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti-inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA-induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP-1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor-kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose-dependent manner. In contrast, phloretin significantly attenuated pro-inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor-kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA-induced endothelial injury via a synergic mechanism including direct anti-inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia-related cardiovascular diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Two Sulfur Glycoside Compounds Isolated from Lepidium apetalum Willd Protect NRK52e Cells against Hypertonic-Induced Adhesion and Inflammation by Suppressing the MAPK Signaling Pathway and RAAS.

PubMed

Yuan, Peipei; Zheng, Xiaoke; Li, Meng; Ke, Yingying; Fu, Yang; Zhang, Qi; Wang, Xiaolan; Feng, Weisheng

2017-11-12

Lepidium apetalum Willd has been used to reduce edema and promote urination. Cis -desulfoglucotropaeolin ( cis -DG) and trans -desulfoglucotropaeolin ( trans -DG) were isolated from Lepidium apetalum Willd, and caused a significant increase in cell viability in a hypertonic model in NRK52e cells. In the hypertonic model, cis -DG and trans -DG significantly promoted the cell viability of NRK52e cells and inhibited the elevation of Na⁺ in the supernatant, inhibited the renin-angiotensin-aldosterone (RAAS) system, significantly reduced the levels of angiotensin II (Ang II) and aldosterone (ALD), and lowered aquaporin-2 (AQP2) and Na⁺-K⁺ ATP content in renal medulla. After treatment with cis -DG and trans -DG, expression of calcineurin (CAN) and Ca/calmodulin-dependent protein kinase II (CaMK II) was decreased in renal tissue and Ca 2+ influx was inhibited, thereby reducing the secretion of transforming growth factor-β (TGFβ), reversing the increase in adhesion and inflammatory factor E-selectin and monocyte chemotactic protein 1 (MCP-1) induced by high NaCl, while reducing oxidative stress status and decreasing the expression of cyclooxygenase-2 (COX2). Furthermore, inhibition of protein kinase C (PKC) expression also contributed to these improvements. The cis -DG and trans -DG reduced the expression of p-p44/42 MAPK, p-JNK and p-p38, inhibited the phosphorylation of the MAPK signaling pathway in NRN52e cells induced by high salt, decreased the overexpression of p-p38 and p-HSP27, and inhibited the overactivation of the p38-MAPK signaling pathway, suggesting that the p38-MAPK pathway may play a vital role in the hypertonic-induced adhesion and inflammatory response. From the results of this study, it can be concluded that the mechanism of cis -DG and trans -DG may mainly be through inhibiting the p38-MAPK signaling pathway, inhibiting the excessive activation of the RAAS system, and thereby reducing adhesion and inflammatory factors.

Hyaluronic Acid-Serum Hydrogels Rapidly Restore Metabolism of Encapsulated Stem Cells and Promote Engraftment

PubMed Central

Chan, Angel T.; Karakas, Mehmet F.; Vakrou, Styliani; Afzal, Junaid; Rittenbach, Andrew; Lin, Xiaoping; Wahl, Richard L.; Pomper, Martin G.; Steenbergen, Charles J.; Tsui, Benjamin M.W.; Elisseeff, Jennifer H.; Abraham, M. Roselle

2015-01-01

Background Cell death due to anoikis, necrosis and cell egress from transplantation sites limits functional benefits of cellular cardiomyoplasty. Cell dissociation and suspension, which are a pre-requisite for most cell transplantation studies, lead to depression of cellular metabolism and anoikis, which contribute to low engraftment. Objective We tissue engineered scaffolds with the goal of rapidly restoring metabolism, promoting viability, proliferation and engraftment of encapsulated stem cells. Methods The carboxyl groups of HA were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA-NHS) groups that react with free amine groups to form amide bonds. HA-NHS was cross-linked by serum to generate HA:Serum (HA:Ser) hydrogels. Physical properties of HA:Ser hydrogels were measured. Effect of encapsulating cardiosphere-derived cells (CDCs) in HA:Ser hydrogels on viability, proliferation, glucose uptake and metabolism was assessed in vitro. In vivo acute intra-myocardial cell retention of 18FDG-labeled CDCs encapsulated in HA:Ser hydrogels was quantified. Effect of CDC encapsulation in HA:Ser hydrogels on in vivo metabolism and engraftment at 7 days was assessed by serial, dual isotope SPECT-CT and bioluminescence imaging of CDCs expressing the Na-iodide symporter and firefly luciferase genes respectively. Effect of HA:Ser hydrogels +/− CDCs on cardiac function was assessed at 7 days & 28 days post-infarct. Results HA:Ser hydrogels are highly bio-adhesive, biodegradable, promote rapid cell adhesion, glucose uptake and restore bioenergetics of encapsulated cells within 1 h of encapsulation, both in vitro and in vivo. These metabolic scaffolds can be applied epicardially as a patch to beating hearts or injected intramyocardially. HA:Ser hydrogels markedly increase acute intramyocardial retention (~6 fold), promote in vivo viability, proliferation, engraftment of encapsulated stem cells and angiogenesis. Conclusion HA:Ser hydrogels serve as ‘synthetic stem cell niches’ that rapidly restore metabolism of encapsulated stem cells, promote stem cell engraftment and angiogenesis. These first ever, tissue engineered metabolic scaffolds hold promise for clinical translation in conjunction with CDCs and possibly other stem cell types. PMID:26378976

Study of probiotic potential of four wild Lactobacillus rhamnosus strains.

PubMed

Tuo, Yanfeng; Zhang, Weiqin; Zhang, Lanwei; Ai, Lianzhong; Zhang, Yingchun; Han, Xue; Yi, Huaxi

2013-06-01

The four wild Lactobacillus rhamnosus strains were examined in vitro for resistance to simulated gastro and intestinal juices, adhesion to HT-29 cells, antagonistic activity against enteric pathogens and immunomodulating activity. The strains L. rhamnosus SB5L, J5L and IN1L were able to survive in simulated gastro juice while the strain L. rhamnosus SB31L lost viability exposed to simulated gastro juice for 3 h. The four strains had high viability in simulated small intestinal juice with little loss (<1.0 cycle reduction). The strains SB5L, J5L and IN1L antagonized against Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028, Shigella sonnei ATCC 25931. The strain L. rhamnosus IN1L had the highest adhesive capability to HT-29 cells in vitro (251 bacteria cells per 100 HT-29 cells) compared to the other three L. rhamnosus strains. The live bacteria, cell wall and DNA of the four L. rhamnosus induced the secretion of pro-inflammatory cytokines IL-12 (p70), IFN-γ and TNF-α by human peripheral blood mononuclear cells (PBMCs). The levels of IL-12 (p70), IFN-γ and TNF-α produced by stimulated PBMCs were significantly higher (P < 0.05) than those of the control. Those data indicated that the four L. rhamnosus strains have the potential as the probiotic for human being use, although further studies are still needed. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

PubMed Central

Burkholder, Kristin M; Bhunia, Arun K

2009-01-01

Background Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) to Caco-2 cells exposed to thermal stress (41°C, 1 h) was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of Lactobacillus rhamnosus GG and Lactobacillus gasseri on Salmonella adhesion and Salmonella-induced cytotoxicity of Caco-2 cells subjected to thermal stress. Results Thermal stress increased the cytotoxic effect of both S. Typhimurium (P = 0.0001) and nonpathogenic E. coli K12 (P = 0.004) to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to S. Typhimurium adhesion (P = 0.001). Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to S. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking S. Typhimurium with anti-ShdA antibody prior to exposure of Salmonella decreased adhesion (P = 0.01) to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to L. rhamnosus GG significantly reduced Salmonella-induced cytotoxicity (P = 0.001) and Salmonella adhesion (P = 0.001) to Caco-2 cells during thermal stress, while L. gasseri had no effect. Conclusion Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to Salmonella adhesion, and increases the cytotoxic effect of Salmonella during infection. Use of L. rhamnosus GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms by which thermal stress increases susceptibility to S. Typhimurium colonization and by which L. rhamnosus GG limits the severity of infection remain to be elucidated. PMID:19589170

Chitosan-catechol: a writable bioink under serum culture media.

PubMed

Lee, Daiheon; Park, Joseph P; Koh, Mi-Young; Kim, Pureum; Lee, Junhee; Shin, Mikyung; Lee, Haeshin

2018-05-01

Mussel-inspired adhesive coatings on biomedical devices have attracted significant interest due to their unique properties such as substrate independency and high efficiency. The key molecules for mussel-inspired adhesive coatings are catechol and amine groups. Along with the understanding of catechol chemistry, chitosan-catechol has also been developed as a representative mussel-inpired adhesive polymer that contains catechol and amine groups for adhesiveness. Herein, we demonstrated the direct writability of chitosan-catechol as a bioink for 3D printing, one of the additive techniques. The use of chitosan-catechol bioink results in the formation of 3D constructs in normal culture media via rapid complexation of this bioink with serum proteins; in addition, the metal/catechol combination containing tiny amounts of vanadyl ions, in which the ratio of metal to catechol is 0.0005, dramatically enhances the mechanical strength and printability of the cell-encapsulated inks, showing a cell viability of approximately 90%. These findings for mussel-inspired bioinks will be a promising way to design a biocompatible 3D bioink cross-linked without any external stimuli.

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear

PubMed Central

Aliuos, Pooyan; Schulze, Jennifer; Schomaker, Markus; Reuter, Günter; Stolle, Stefan R. O.; Werner, Darja; Ripken, Tammo; Lenarz, Thomas; Warnecke, Athanasia

2016-01-01

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest. PMID:26918945

Dual antibacterial agents of nano-silver and 12-methacryloyloxydodecylpyridinium bromide in dental adhesive to inhibit caries

PubMed Central

Zhang, Ke; Li, Fang; Imazato, Satoshi; Cheng, Lei; Liu, Huaibing; Arola, Dwayne D.; Bai, Yuxing; Xu, Hockin H. K.

2013-01-01

Dental resins containing 12-methacryloyloxydodecylpyridinium bromide (MDPB) showed potent antibacterial functions. Recent studies developed antibacterial resins containing nanoparticles of silver (NAg). The objectives of this study were to develop an adhesive containing dual agents of MDPB and NAg for the first time, and to investigate the combined effects of antibacterial adhesive and primer on biofilm viability, metabolic activity, lactic acid, dentin bond strength, and fibroblast cytotoxicity. MDPB and NAg were incorporated into Scotchbond Multi-Purpose (SBMP) adhesive “A” and primer “P”. Five systems were tested: SBMP adhesive A; A+MDPB; A+NAg; A+MDPB+NAg; P+MDPB+NAg together with A+MDPB+NAg. Dental plaque microcosm biofilms were cultured using mixed saliva from ten donors. Metabolic activity, colony-forming units, and lactic acid production of biofilms were investigated. Human fibroblast cytotoxicity of bonding agents was determined. MDPB+NAg in adhesive/primer did not compromise dentin bond strength (p>0.1). MDPB or NAg alone in adhesive substantially reduced the biofilm activities. Dual agents MDPB+NAg in adhesive greatly reduced the biofilm viability compared to each agent alone (p<0.05). The greatest inhibition of biofilms was achieved when both adhesive and primer contained MDPB+NAg. Fibroblast viability of groups with dual antibacterial agents was similar to control using culture medium without resin eluents (p>0.1). In conclusion, this study showed for the first time that the antibacterial potency of MDPB adhesive could be substantially enhanced via NAg. Adding MDPB+NAg into both primer and adhesive achieved the strongest anti-biofilm efficacy. The dual agent (MDPB+NAg) method could have wide applicability to other adhesives, sealants, cements and composites to inhibit biofilms and caries. PMID:23529901

Endothelial cell behaviour on gas-plasma-treated PLA surfaces: the roles of surface chemistry and roughness.

PubMed

Shah, Amita; Shah, Sarita; Mani, Gopinath; Wenke, Joseph; Agrawal, Mauli

2011-04-01

Glow-discharge gas-plasma (GP) treatment has been shown to induce surface modifications such that cell adhesion and growth are enhanced. However, it is not known which gas used in GP treatment is optimal for endothelial cell function. Polylactic acid (PLA) films treated oxygen, argon, or nitrogen GP were characterized using contact angles, scanning electron microscopy, atomic force microscopy, optical profilometry, and x-ray photoelectron spectroscopy. All three GP treatments decreased the carbon atomic concentration and surface roughness and increased the oxygen atomic concentration. Human umbilical vein endothelial cells were cultured on the PLA films for up to 7 days. Based on proliferation and live/dead assays, surface chemistry was shown to have the greatest effect on the attachment, proliferation, and viability of these cells, while roughness did not have a significant influence. Of the different gases, endothelial cell viability, attachment and proliferation were most significantly increased on PLA surfaces treated with oxygen and argon gas plasma. Copyright © 2010 John Wiley & Sons, Ltd.

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

PubMed Central

Vyas, Sejal; Chesarone-Cataldo, Melissa; Todorova, Tanya; Huang, Yun-Han; Chang, Paul

2013-01-01

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD+ as their substrate to modify acceptor proteins with adenosine diphosphate-ribose (ADPr) modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyze the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knock-down phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose), and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division, and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology. PMID:23917125

Preparation and characterization of a photocrosslinkable bioadhesive inspired by marine mussel.

PubMed

Xue, Jie; Wang, Tao; Nie, Jun; Yang, Dongzhi

2013-02-05

Synthetic adhesives inspired by marine mussel have attracted great attention due to its excellent water-resistance and good biocompatibility. In this study, a photocrosslinkable bioadhesive containing 3,4-Dihydroxy-l-phenylalanine (DOPA) functional group, which is central to curing mussel adhesive proteins, was prepared by ultraviolet (UV) irradiation of a new photocurable monomer ethylene glycol acrylate methacrylate-dopamine (EGAMA-DOPA) and a UV photocrosslinkable crosslinking agent poly(vinyl alcohol) (UV-PVA) derivative. The chemical structures of EGAMA-DOPA and UV-PVA were confirmed by Fourier Transform Infrared Reflection (FTIR) and (1)H NMR spectroscopy, respectively. The effects of UV light intensity, content of photoinitiator, EGAMA-DOPA and UV-PVA on the photopolymerization kinetics were studied, and the effects of the content of UV-PVA and temperature on the adhesive strength were also investigated. It was found that the higher UV light intensity, the faster polymerization rate and the higher final conversion that was the same as the trend of photoinitiator, EGAMA-DOPA and UV-PVA. And the adhesion strength measurement showed that, for gels with 30wt.% EGAMA-DOPA, the adhesion strength was obviously improved by about 150% with 3.0wt.% UV-PVA instead of pure PVA, and for gels containing 40wt.% EGAMA-DOPA, the adhesion strength sharply enhanced by 123% with increasing the content of UV-PVA from 1.0wt.% to 3.0wt.%. Cell attachment results showed good cell viability of L929 cell on the EGAMA-DOPA/UV-PVA adhesive gels. Thanks to its strong adhesion strength and good biocompatibility, such photocrosslinkable gels could be applied to the areas of biomedical field. Copyright © 2012 Elsevier B.V. All rights reserved.

MDA-MB-231 breast cancer cell viability, motility and matrix adhesion are regulated by a complex interplay of heparan sulfate, chondroitin-/dermatan sulfate and hyaluronan biosynthesis.

PubMed

Viola, Manuela; Brüggemann, Kathrin; Karousou, Evgenia; Caon, Ilaria; Caravà, Elena; Vigetti, Davide; Greve, Burkhard; Stock, Christian; De Luca, Giancarlo; Passi, Alberto; Götte, Martin

2017-06-01

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

PubMed Central

Bou Zeidan, Marc; Carmona, Lourdes

2013-01-01

Saccharomyces cerevisiae “flor” yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends on FLO11 gene length and expression. FLO11 encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm of S. cerevisiae flor strains. We found that PAF26, a synthetic tryptophan-rich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functional FLO11 but was not accompanied by increased FLO11 expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that a flo11 gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that the FLO11 gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-to-cell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides. PMID:23892742

A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwata, Futoshi, E-mail: iwata.futoshi@shizuoka.ac.jp; Research Institute of Electronics, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8011; Adachi, Makoto

We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells wasmore » evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.« less

Hydrogen sulfide facilities production of nitric oxide via the Akt/endothelial nitric oxide synthases signaling pathway to protect human umbilical vein endothelial cells from injury by angiotensin II.

PubMed

Cui, Jiasen; Zhuang, Shunjiu; Qi, Shaohong; Li, Li; Zhou, Junwen; Zhang, Wan; Zhao, Yun; Qi, Ning; Yin, Yangjun; Huang, Lu

2017-11-01

Angiotensin II (Ang II) has been reported as key in inducing endothelial cell injury, and endothelial cells may produce nitric oxide (NO) to protect themselves. However, the underlying mechanism remains elusive. Human umbilical vein endothelial cells (HUVECs) were divided into five treatment groups as follows: Normal control, Ang II, Ang II + sodium hydrosulfide [NaHS; hydrogen sulfide (H2S) donor], Ang II + Akt inhibitors + NaHS, and Ang II + endothelial nitric oxide synthases (eNOS) inhibitors + NaHS. Subsequently, cell viability, apoptosis, migration, proliferation and adhesion ability were determined. In addition, tubular structure formation was observed, and the NO and phosphorylation levels of Akt and eNOS were evaluated. Compared with the normal control group, Ang II treatment reduced the viability of HUVECs and increased the level of cell apoptosis (P<0.05). Furthermore, Ang II treatment inhibited the phosphorylation level of eNOS and Akt, as well as the generation of NO (P<0.05). H2S reversed the above‑mentioned effects significantly and increased cell proliferation, adhesion ability and promoted tubular structure formation (P<0.05); however, H2S did not reverse the impact of eNOS and Akt phosphorylation levels after being processed with Akt and eNOS inhibitors, which indicates that H2S is capable of protecting HUVECs via the eNOS/Akt signaling pathway (P<0.05). Thus, H2S stimulates the production of NO and protects HUVECs via inducing the Akt/eNOS signaling pathway.

Baicalein inhibits the migration and invasive properties of human hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiu, Yung-Wei; Institute of Medicine, Chung Shan Medical University, Taiwan; Lin, Tseng-Hsi

Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24 h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38more » mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-{beta}. In addition, baicalein reduced the phosphorylation levels of PKC{alpha} and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo. - Highlight: > Baicalein inhibits several essential steps in the onset of metastasis.« less

Effect of testosterone incorporation on cell proliferation and differentiation for polymer-bioceramic composites.

PubMed

da Costa, Kelen Jorge Rodrigues; Passos, Joel J; Gomes, Alinne D M; Sinisterra, Rubén D; Lanza, Célia R M; Cortés, Maria Esperanza

2012-11-01

In the current study, we characterized the polycaprolactone (PCL), poly(lactic acid-co-glycolic acid) (PLGA), and biphasic calcium phosphate (BCP) composites coated with testosterone propionate (T) using Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (XRD). Osteoblastic cells were seeded with PCL/BCP, PCL/BCP/T, PLGA/PCL/BCP and PLGA/PCL/BCP/T scaffolds, and cell viability, proliferation, differentiation and adhesion were analyzed. The results of physic-chemical experiments showed no displacements or suppression of bands in the FTIR spectra of scaffolds. The XRD patterns of the scaffolds showed an amorphous profile. The osteoblastic cells viability and proliferation increased in the presence of composites with testosterone over 72 h, and were significantly greater when PLGA/PCL/BCP/T scaffold was tested against PCL/BCP/T. Furthermore alkaline phosphatase production was significantly greater in the same group. In conclusion, the PLGA/PCL/BCP scaffold with testosterone could be a promising option for bone tissue applications due to its biocompatibility and its stimulatory effect on cell proliferation.

The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sepulveda-Medina, Paola; Katsenovich, Yelena; Musaramthota, Vishal

Nuclear production facilities during the Cold War have caused liquid waste to leak and soak into the ground creating multiple radionuclide plumes. The Arthrobacter bacteria are one of the most common groups in soils and are found in large numbers in subsurface environments contaminated with radionuclides. This study experimentally analyzed changes on the bacteria surface after uranium exposure and evaluated the effect of bicarbonate ions on U(VI) toxicity of a less uranium tolerant Arthrobacter strain, G968, by investigating changes in adhesion forces and cells dimensions via atomic force microscopy (AFM). AFM and viability studies showed that samples containing bicarbonate aremore » able to acclimate and withstand uranium toxicity. Samples containing no bicarbonate exhibited deformed surfaces and a low height profile, which might be an indication that the cells are not alive.« less

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

NASA Astrophysics Data System (ADS)

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V.; Noll, Thomas; Funk, Richard H. W.; Engelmann, Katrin; Werner, Carsten

2015-08-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

PubMed

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V; Noll, Thomas; Funk, Richard H W; Engelmann, Katrin; Werner, Carsten

2015-08-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly( N -isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na + /K + -ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro . The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

PubMed Central

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V; Noll, Thomas; Funk, Richard H W; Engelmann, Katrin; Werner, Carsten

2015-01-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty. PMID:27877823

Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions

PubMed Central

Tanti, N.C.; Jones, L.; Sheardown, H.

2010-01-01

Purpose Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate  (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Conclusions Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells. PMID:20169012

Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions.

PubMed

Gorbet, M B; Tanti, N C; Jones, L; Sheardown, H

2010-02-19

Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of beta(1) and alpha(3) integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells.

Adhesion force of staphylococcus aureus on various biomaterial surfaces.

PubMed

Alam, Fahad; Balani, Kantesh

2017-01-01

Staphylococcus comprises of more than half of all pathogens in orthopedic implant infections and they can cause major bone infection which can result in destruction of joint and bone. In the current study, adhesion force of bacteria on the surface of various biomaterial surfaces is measured using atomic force microscope (AFM). Staphylococcus aureus was immobilized on an AFM tipless cantilever as a force probe to measure the adhesion force between bacteria and biomaterials (viz. ultra-high molecular weight poly ethylene (UHMWPE), stainless steel (SS), Ti-6Al-4V alloy, hydroxyapatite (HA)). At the contact time of 10s, UHMWPE shows weak adhesion force (~4nN) whereas SS showed strong adhesion force (~15nN) due to their surface energy and surface roughness. Bacterial retention and viability experiment (3M™ petrifilm test, agar plate) dictates that hydroxyapatite shows the lowest vaibility of bacteria, whereas lowest bacterial retention is observed on UHMWPE surface. Similar results were obtained from live/dead staining test, where HA shows 65% viability, whereas on UHMWPE, SS and Ti-6Al-4V, the bacterial viability is 78%, 94% and 97%, respectively. Lower adhesion forces, constrained pull-off distance (of bacterial) and high antibacterial resistance of bioactive-HA makes it a potential biomaterial for bone-replacement arthroplasty. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aspirin, lysine, mifepristone and doxycycline combined can effectively and safely prevent and treat cancer metastasis: prevent seeds from gemmating on soil

PubMed Central

Xu, Huo; Ma, Ji; Zhu, Yewei; Lu, Yusheng; Wang, Jichuang; Zhang, Ting; Li, Tao; Xie, Jingjing; Xu, Bo; Xie, Fangwei; Gao, Yu; Shao, Jingwei; Tu, Xiaohuang; Jia, Lee

2015-01-01

Recent scientific advances have increased our understanding of the cancer metastatic complexities and provided further impetus for new combination therapies to prevent cancer metastasis. Here, we demonstrated that a combination (HAMPT) of aspirin, lysine, mifepristone and doxycycline can effectively and safely prevent cancer metastasis. The pharmaceutically-formulated HAMPT inhibited adhesion of cancer cells to either endothelial cells or extracellular matrix via down-regulating cell adhesion molecules ICAM-1 and α4-integrin. HAMPT inhibited the cloak effect by activated platelets on cancer cells, thereby interfering adhesion and invasion of cancer cells to the underlying stroma. At the effective concentration, HAMPT induced cancer cells into dormancy with minor inhibition on cell viability. Four-day pretreatment followed by 30-day oral administration of HAMPT (33.5-134 mg/kg) to the mice inoculated with cancer cells produced significant inhibition on cancer metastasis dose-dependently without marked side effects. Fifty-day rat toxicity study with HAMPT at doses (335-1340 mg/kg) 20-fold higher than its therapeutic dose produced no significant toxicity. Interestingly, the acute toxic death could not be reached at the maximum administrable dose (5 g/kg). This proof-of-concept study provides the first conceptual evidence that cancer metastasis can be controlled by using affordable old drugs to restrain circulating tumor cells from gemmating on the metastatic soil without the need for cytotoxicity. PMID:26459390

Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

PubMed Central

Sofrenovic, Tanja; McEwan, Kimberly; Crowe, Suzanne; Marier, Jenelle; Davies, Robbie; Suuronen, Erik J.; Kuraitis, Drew

2012-01-01

Background Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs). Methods PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential. Results The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34+VEGFR2+CD133+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle. Conclusion Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34+VEGFR2+CD133+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation. PMID:23133548

Electrospun Fe3O4/TiO2 hybrid nanofibers and their in vitro biocompatibility: prospective matrix for satellite cell adhesion and cultivation.

PubMed

Amna, Touseef; Hassan, M Shamshi; Van Ba, Hoa; Khil, Myung-Seob; Lee, Hak-Kyo; Hwang, I H

2013-03-01

We report the fabrication of novel Fe3O4/TiO2 hybrid nanofibers with the improved cellular response for potential tissue engineering applications. In this study, Fe3O4/TiO2 hybrid nanofibers were prepared by facile sol-gel electrospinning using titanium isopropoxide and iron(III) nitrate nonahydrate as precursors. The obtained electrospun nanofibers were vacuum dried at 80 °C and then calcined at 500 °C. The physicochemical characterization of the synthesized composite nanofibers was carried out by scanning electron microscopy, energy dispersive X-ray spectroscopy, transmission electron microscopy and X-ray diffraction pattern. To examine the in vitro cytotoxicity, satellite cells were treated with as-prepared Fe3O4/TiO2 and the viability of cells was analyzed by Cell Counting Kit-8 assay at regular time intervals. The morphological features of unexposed satellite cells and exposed to Fe3O4/TiO2 composite were examined with a phase contrast microscope whereas the quantification of cell viability was carried out via confocal laser scanning microscopy. The morphology of the cells attached to hybrid matrix was observed by Bio-SEM. Cytotoxicity experiments indicated that the satellite cells could attach to the Fe3O4/TiO2 composite nanofibers after being cultured. We observed that Fe3O4-TiO2 composite nanofibers could support cell adhesion and growth. Results from this study therefore suggest that Fe3O4/TiO2 composite scaffold with small diameters (approximately 200 nm) can mimic the natural extracellular matrix well and provide possibilities for diverse applications in the field of tissue engineering and regenerative medicine. Copyright © 2012 Elsevier B.V. All rights reserved.

In vitro and in vivo evaluation of a new nanocomposite, containing high density polyethylene, tricalcium phosphate, hydroxyapatite, and magnesium oxide nanoparticles.

PubMed

Pourdanesh, Fereydoun; Jebali, Ali; Hekmatimoghaddam, Seyedhossein; Allaveisie, Azra

2014-07-01

In this study, a new nanocomposite, which contained high density polyethylene (HDPE), tricalcium phosphate (Ca3(PO4)2) nanoparticles (TCP NPs), hydroxyapatite nanoparticles (HA NPs), and magnesium oxide nanoparticles (MgO NPs) was prepared. As in vitro experiment, human osteoblasts (HOB) cells were exposed to pristine HDPE and its nanocomposite for a period of 1, 4, and 7 days at 37 °C, and then different assays were carried out, including osteoblast cell proliferation, Trypan blue staining, cell viability, alkaline phosphatase (ALP), and cell adhesion. Antibacterial property of pristine HDPE and its nanocomposite was evaluated, and also their mechanical properties were measured after 2 and 4 months. As in vivo experiment, pristine HDPE and its nanocomposite were separately implanted on calvarium bone of rabbits, and tissue inflammation and osteogenesis were investigated after 2, 4, and 6 months. In case of HOB cells treated with HDPE or nanocomposite, as incubation time was increased, cell proliferation, live/dead ratio, and cell viability were decreased. But, the ALP activity and cell adhesion of HOB cells which treated with nanocomposite were raised after increase of incubation time. This study demonstrated that although the mechanical properties of nanocomposite were similar to HDPE sheet, but their antibacterial property was not similar. The in vivo experiment showed that both pristine HDPE and its nanocomposite had same inflammation responses. Interestingly, osteogenesis was observed after 2 months at bone/nanocomposite interface, and was highly increased after 4 and 6 months. It must be noted that such pattern was not seen at bone/HDPE interface. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro study of histamine and histamine receptor ligands influence on the adhesion of purified human eosinophils to endothelium.

PubMed

Grosicki, Marek; Wójcik, Tomasz; Chlopicki, Stefan; Kieć-Kononowicz, Katarzyna

2016-04-15

It is a well-known fact that histamine is involved in eosinophil-dependent inflammatory responses including cellular chemotaxis and migration. Nevertheless, the relative role of histamine receptors in the mechanisms of eosinophils adhesion to endothelial cells is not known. Therefore the aim of presented study was to examine the effect of selective histamine receptors ligands on eosinophils adhesion to endothelium. For that purpose the highly purified human eosinophils have been isolated from the peripheral blood. The viability and functional integrity of isolated eosinophils have been validated in several tests. Histamine as well as 4-methylhistamine (selective H4 agonist) in concentration-dependent manner significantly increased number of eosinophils that adhere to endothelium. Among the selective histamine receptors antagonist or H1 inverse agonist only JNJ7777120 (histamine H4 antagonist) and thioperamide (dual histamine H3/H4 antagonist) had direct effect on eosinophils adhesion to endothelial cells. Antagonists of H1 (diphenhydramine, mepyramine) H2 (ranitidine and famotidine) and H3 (pitolisant) histamine receptors were ineffective. To the best of our knowledge, this is the first study to demonstrate that histamine receptor H4 plays a dominant role in histamine-induced eosinophils adhesion to endothelium. Copyright © 2016 Elsevier B.V. All rights reserved.

Photocrosslinkable Gelatin/Tropoelastin Hydrogel Adhesives for Peripheral Nerve Repair.

PubMed

Soucy, Jonathan R; Shirzaei Sani, Ehsan; Portillo Lara, Roberto; Diaz, David; Dias, Felipe; Weiss, Anthony S; Koppes, Abigail N; Koppes, Ryan A; Annabi, Nasim

2018-05-09

Suturing peripheral nerve transections is the predominant therapeutic strategy for nerve repair. However, the use of sutures leads to scar tissue formation, hinders nerve regeneration, and prevents functional recovery. Fibrin-based adhesives have been widely used for nerve reconstruction, but their limited adhesive and mechanical strength and inability to promote nerve regeneration hamper their utility as a stand-alone intervention. To overcome these challenges, we engineered composite hydrogels that are neurosupportive and possess strong tissue adhesion. These composites were synthesized by photocrosslinking two naturally derived polymers, gelatin-methacryloyl (GelMA) and methacryloyl-substituted tropoelastin (MeTro). The engineered materials exhibited tunable mechanical properties by varying the GelMA/MeTro ratio. In addition, GelMA/MeTro hydrogels exhibited 15-fold higher adhesive strength to nerve tissue ex vivo compared to fibrin control. Furthermore, the composites were shown to support Schwann cell (SC) viability and proliferation, as well as neurite extension and glial cell participation in vitro, which are essential cellular components for nerve regeneration. Finally, subcutaneously implanted GelMA/MeTro hydrogels exhibited slower degradation in vivo compared with pure GelMA, indicating its potential to support the growth of slowly regenerating nerves. Thus, GelMA/MeTro composites may be used as clinically relevant biomaterials to regenerate nerves and reduce the need for microsurgical suturing during nerve reconstruction.

Adhesion, Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

PubMed Central

Strauß, Sarah; Neumeister, Anne; Barcikowski, Stephan; Kracht, Dietmar; Kuhbier, Jörn W.; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M.

2013-01-01

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells. PMID:23308190

Evaluation of cell responses toward adhesives with different photoinitiating systems.

PubMed

Van Landuyt, Kirsten L; Krifka, Stephanie; Hiller, Karl-Anton; Bolay, Carola; Waha, Claudia; Van Meerbeek, Bart; Schmalz, Gottfried; Schweikl, Helmut

2015-08-01

The photoinitiator diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) is more reactive than a camphorquinone/amine (CQ) system, and TPO-based adhesives obtained a higher degree of conversion (DC) with fewer leached monomers. The hypothesis tested here is that a TPO-based adhesive is less toxic than a CQ-based adhesive. A CQ-based adhesive (SBU-CQ) (Scotchbond Universal, 3M ESPE) and its experimental counterpart with TPO (SBU-TPO) were tested for cytotoxicity in human pulp-derived cells (tHPC). Oxidative stress was analyzed by the generation of reactive oxygen species (ROS) and by the expression of antioxidant enzymes. A dentin barrier test (DBT) was used to evaluate cell viability in simulated clinical circumstances. Unpolymerized SBU-TPO was significantly more toxic than SBU-CQ after a 24h exposure, and TPO alone (EC50=0.06mM) was more cytotoxic than CQ (EC50=0.88mM), EDMAB (EC50=0.68mM) or CQ/EDMAB (EC50=0.50mM). Cultures preincubated with BSO (l-buthionine sulfoximine), an inhibitor of glutathione synthesis, indicated a minor role of glutathione in cytotoxic responses toward the adhesives. Although the generation of ROS was not detected, a differential expression of enzymatic antioxidants revealed that cells exposed to unpolymerized SBU-TPO or SBU-CQ are subject to oxidative stress. Polymerized SBU-TPO was more cytotoxic than SBU-CQ under specific experimental conditions only, but no cytotoxicity was detected in a DBT with a 200μm dentin barrier. Not only DC and monomer-release determine the biocompatibility of adhesives, but also the cytotoxicity of the (photo-)initiator should be taken into account. Addition of TPO rendered a universal adhesive more toxic compared to CQ; however, this effect could be annulled by a thin dentin barrier. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Both Leukotoxin and Poly-N-Acetylglucosamine Surface Polysaccharide Protect Aggregatibacter actinomycetemcomitans Cells from Macrophage Killing

PubMed Central

Venketaraman, Vishwanath; Lin, Albert K.; Le, Amy; Kachlany, Scott C.; Connell, Nancy D.; Kaplan, Jeffrey B.

2008-01-01

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately 2-fold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans. PMID:18573331

Targeting the yin and the yang: combined inhibition of the tyrosine kinase c-Src and the tyrosine phosphatase SHP-2 disrupts pancreatic cancer signaling and biology in vitro and tumor formation in vivo.

PubMed

Gomes, Evan G; Connelly, Sarah F; Summy, Justin M

2013-07-01

Although c-Src (Src) has emerged as a potential pancreatic cancer target in preclinical studies, Src inhibitors have not demonstrated a significant therapeutic benefit in clinical trials. The objective of these studies was to examine the effects of combining Src inhibition with inhibition of the protein tyrosine phosphatase SHP-2 in pancreatic cancer cells in vitro and in vivo. SHP-2 and Src functions were inhibited by siRNA or small molecule inhibitors. The effects of dual Src/SHP-2 functional inhibition were evaluated by Western blot analysis of downstream signaling pathways; cell biology assays to examine caspase activity, viability, adhesion, migration, and invasion in vitro; and an orthotopic nude mouse model to observe pancreatic tumor formation in vivo. Dual targeting of Src and SHP-2 induces an additive or supra-additive loss of phosphorylation of Akt and ERK-1/2 and corresponding increases in expression of apoptotic markers, relative to targeting either protein individually. Combinatorial inhibition of Src and SHP-2 significantly reduces viability, adhesion, migration, and invasion of pancreatic cancer cells in vitro and tumor formation in vivo, relative to individual Src/SHP-2 inhibition. These data suggest that the antitumor effects of Src inhibition in pancreatic cancer may be enhanced through simultaneous inhibition of SHP-2.

Osteoselection supported by phase separated polymer blend films.

PubMed

Gulsuner, Hilal Unal; Gengec, Nevin Atalay; Kilinc, Murat; Erbil, H Yildirim; Tekinay, Ayse B

2015-01-01

The instability of implants after placement inside the body is one of the main obstacles to clinically succeed in periodontal and orthopedic applications. Adherence of fibroblasts instead of osteoblasts to implant surfaces usually results in formation of scar tissue and loss of the implant. Thus, selective bioadhesivity of osteoblasts is a desired characteristic for implant materials. In this study, we developed osteoselective and biofriendly polymeric thin films fabricated with a simple phase separation method using either homopolymers or various blends of homopolymers and copolymers. As adhesive and proliferative features of cells are highly dependent on the physicochemical properties of the surfaces, substrates with distinct chemical heterogeneity, wettability, and surface topography were developed and assessed for their osteoselective characteristics. Surface characterizations of the fabricated polymer thin films were performed with optical microscopy and SEM, their wettabilities were determined by contact angle measurements, and their surface roughness was measured by profilometry. Long-term adhesion behaviors of cells to polymer thin films were determined by F-actin staining of Saos-2 osteoblasts, and human gingival fibroblasts, HGFs, and their morphologies were observed by SEM imaging. The biocompatibility of the surfaces was also examined through cell viability assay. Our results showed that heterogeneous polypropylene polyethylene/polystyrene surfaces can govern Saos-2 and HGF attachment and organization. Selective adhesion of Saos-2 osteoblasts and inhibited adhesion of HGF cells were achieved on micro-structured and hydrophobic surfaces. This work paves the way for better control of cellular behaviors for adjustment of cell material interactions. © 2014 Wiley Periodicals, Inc.

Modulation of cell adhesion and viability of cultured murine bone marrow cells by arsenobetaine, a major organic arsenic compound in marine animals.

PubMed

Sakurai, T; Fujiwara, K

2001-01-01

1. In this study, we investigated the biological effects of trimethyl (carboxymethyl) arsonium zwitterion, namely arsenobetaine (AsBe), which is a major organic arsenic compound in marine animals using murine bone marrow (BM) cells and compared them with those of an inorganic arsenical, sodium arsenite, in vitro. 2. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC(50) was 6 microM. In contrast, AsBe significantly enhanced the viability of BM cells in a dose-dependent manner during a 72-h incubation; about a twofold increase in the viability of cells compared with that of control cells cultured with the medium alone was observed with a microM level of AsBe. 3. In morphological investigations, AsBe enhanced the numbers of large mature adherent cells, especially granulocytes, during a 72-h BM culture. When BM cells were cultured together with AsBe and a low dose (1 u ml(-1)) of recombinant murine granulocyte/macrophage colony-stimulating factor (rMu GM-CSF), significant additive-like increasing effects were observed on the numbers of both granulocytes and macrophages originated from BM cells. However, AsBe did not cause proliferation of BM cells at all as determined by colony-forming assay using a gelatinous medium. 4. These findings demonstrate the unique and potent biological effects in mammalian cells of AsBe, a major organic arsenic compound in various marine animals which are ingested daily as seafood in many countries.

Phenotype of hepatocyte spheroids in Arg-GLY-Asp (RGD) containing a thermo-reversible extracellular matrix.

PubMed

Park, Keun-Hong; Bae, You Han

2002-07-01

The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.

3D Printed Structures Filled with Carbon Fibers and Functionalized with Mesenchymal Stem Cell Conditioned Media as In Vitro Cell Niches for Promoting Chondrogenesis.

PubMed

García-Ruíz, Josefa Predestinación; Díaz Lantada, Andrés

2017-12-24

In this study, we present a novel approach towards the straightforward, rapid, and low-cost development of biomimetic composite scaffolds for tissue engineering strategies. The system is based on the additive manufacture of a computer-designed lattice structure or framework, into which carbon fibers are subsequently knitted or incorporated. The 3D-printed lattice structure acts as support and the knitted carbon fibers perform as driving elements for promoting cell colonization of the three-dimensional construct. A human mesenchymal stem cell (h-MSC) conditioned medium (CM) is also used for improving the scaffold's response and promoting cell adhesion, proliferation, and viability. Cell culture results-in which scaffolds become buried in collagen type II-provide relevant information regarding the viability of the composite scaffolds used and the prospective applications of the proposed approach. In fact, the advanced composite scaffold developed, together with the conditioned medium functionalization, constitutes a biomimetic stem cell niche with clear potential, not just for tendon and ligament repair, but also for cartilage and endochondral bone formation and regeneration strategies.

Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

NASA Astrophysics Data System (ADS)

Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed by the higher level of cytokines in the medium and elevated share of CD144, ICAM-1 and VCAM-1-positive cells. Comparative analysis of the level TNF-induced secretion of IL-6 and IL-8, as well as the share of cells bearing ICAM-1 and VCAM-1, showed significant variability depending on the donors. Simultaneous exposure to simulated microgravity and proinflammatory activation did not potentiate and did not cancel the effect caused by TNF-a. In summary, our findings indicate that the simulated microgravity is not activating and additional pro-inflammatory stimulus to HUVEC in vitro model. This work was supported in part by Grant from RFBR No.12-04-31763 and Grant No.NSh-371.2014.4

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus.

PubMed

Brink, M; Todorov, S D; Martin, J H; Senekal, M; Dicks, L M T

2006-04-01

Screening of five bile salt-resistant and low pH-tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk-base on cell viability and adhesion of cells to intestinal mucus. Lactobacillus plantarum 423, Lactobacillus casei LHS, Lactobacillus salivarius 241, Lactobacillus curvatus DF 38 and Pediococcus pentosaceus 34 produced the highest level of antimicrobial activity (12,800 AU ml(-1)) when grown in MRS broth supplemented with 2% (m/v) dextrose. Growth in the presence of Raftilose Synergy1, Raftilose L95 and Raftiline GR did not lead to increased levels of antimicrobial activity. Cells grown in the presence of Raftilose Synergy1 took longer to adhere to intestinal mucus, whilst cells grown in the absence of prebiotics showed a linear rate of binding. A broad range of gram-positive and gram-negative bacteria were inhibited. Dextrose stimulated the production of antimicrobial compounds. Adhesion to intestinal mucus did not increase with the addition of prebiotics. The strains may be incorporated in food supplements for HIV/AIDS patients suffering from gastro-intestinal disorders.

Platelet-Poor Plasma as a Supplement for Fibroblasts Cultured in Platelet-Rich Fibrin

PubMed Central

Karam, Sarah Arangurem; Noronha, Thaís Gioda; Sartori, Letícia Regina Morello; San Martin, Alissa Schmidt; Demarco, Flávio Fernando; Conde, Marcus Cristian Muniz

2017-01-01

The aim of this study was to evaluate the proliferation and adhesion of mesenchymal cells (3T3/NIH) in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with Platelet-Poor Plasma (PPP) in aPlatelet-Rich Fibrin (PRF) scaffold. Human blood was obtained and processed in a centrifuge considering the equation G=1.12xRx(RPM/1000)2 to obtain PRF and PPP.Cell adhesion and maintenance analyses were performed by MTTassays in a 96 well plate withsupplemented DMEM: PPP (90:10) for 24 hours. Besides, the PRF was deposited in a 48 well plate and 10x104 cells were seeded above each PRF (n=3) with 800µl of DMEM: PPP (90:10) and cultured for 7 days. Histological analysis and the immunohistochemical staining for Vimentin were performed. Results were analyzed by one-way ANOVA in Stata12®. A significant decrease (p<0.05) of cells adhesion in relationship to FBSwas observed. However, a similar ability of cell-maintenance for PPP 10% was observed (P>0.05). Fibroblasts culture for 7 days in PRF supplemented with PPP 10% was possible, showing positive staining for Vimentin. Therefore, PPP cell supplementation decreased the initial adhesion of cells but was able to maintain the proliferation of adhered cells and able to support their viability in PRF.It seems that this method has many clinical advantagessince it provides an autologous and natural scaffold with their respective supplement for cell culture by only one process, without using xenogeneic compounds. This could improve the potential of clinical translational therapies based on the use of PRF cultured cells, promoting the regenerative potential for future use in medicine and dentistry. PMID:28827850

Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers.

PubMed

Santo, Vítor E; Babo, Pedro; Amador, Miguel; Correia, Cláudia; Cunha, Bárbara; Coutinho, Daniela F; Neves, Nuno M; Mano, João F; Reis, Rui L; Gomes, Manuela E

2016-06-13

Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.

Biocompatibility of hydrophilic silica-coated CdTe quantum dots and magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Ruan, Jing; Wang, Kan; Song, Hua; Xu, Xin; Ji, Jiajia; Cui, Daxiang

2011-12-01

Fluorescent magnetic nanoparticles exhibit great application prospects in biomedical engineering. Herein, we reported the effects of hydrophilic silica-coated CdTe quantum dots and magnetic nanoparticles (FMNPs) on human embryonic kidney 293 (HEK293) cells and mice with the aim of investigating their biocompatibility. FMNPs with 150 nm in diameter were prepared, and characterized by high-resolution transmission electron microscopy and photoluminescence (PL) spectra and magnetometer. HEK293 cells were cultured with different doses of FMNPs (20, 50, and 100μ g/ml) for 1-4 days. Cell viability and adhesion ability were analyzed by CCK8 method and Western blotting. 30 mice were randomly divided into three groups, and were, respectively, injected via tail vein with 20, 60, and 100 μg FMNPs, and then were, respectively, raised for 1, 7, and 30 days, then their lifespan, important organs, and blood biochemical parameters were analyzed. Results show that the prepared water-soluble FMNPs had high fluorescent and magnetic properties, less than 50 μg/ml of FMNPs exhibited good biocompatibility to HEK293 cells, the cell viability, and adhesion ability were similar to the control HEK293 cells. FMNPs primarily accumulated in those organs such as lung, liver, and spleen. Lung exposed to FMNPs displayed a dose-dependent inflammatory response, blood biochemical parameters such as white blood cell count (WBC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), displayed significant increase when the FMNPs were injected into mice at dose of 100μg. In conclusion, FMNPs exhibit good biocompatibility to cells under the dose of less than 50 μg/ml, and to mice under the dose of less than 2mg/kg body weight. The FMNPs' biocompatibility must be considered when FMNPs are used for in vivo diagnosis and therapy.

Effects of magnolol and honokiol on adhesion, yeast-hyphal transition, and formation of biofilm by Candida albicans.

PubMed

Sun, Lingmei; Liao, Kai; Wang, Dayong

2015-01-01

The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary. The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol. Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans. This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection.

A multidisciplinary approach to study the functional properties of neuron-like cell models constituting a living bio-hybrid system: SH-SY5Y cells adhering to PANI substrate

NASA Astrophysics Data System (ADS)

Caponi, S.; Mattana, S.; Ricci, M.; Sagini, K.; Juarez-Hernandez, L. J.; Jimenez-Garduño, A. M.; Cornella, N.; Pasquardini, L.; Urbanelli, L.; Sassi, P.; Morresi, A.; Emiliani, C.; Fioretto, D.; Dalla Serra, M.; Pederzolli, C.; Iannotta, S.; Macchi, P.; Musio, C.

2016-11-01

A living bio-hybrid system has been successfully implemented. It is constituted by neuroblastic cells, the SH-SY5Y human neuroblastoma cells, adhering to a poly-anyline (PANI) a semiconductor polymer with memristive properties. By a multidisciplinary approach, the biocompatibility of the substrate has been analyzed and the functionality of the adhering cells has been investigated. We found that the PANI films can support the cell adhesion. Moreover, the SH-SY5Y cells were successfully differentiated into neuron-like cells for in vitro applications demonstrating that PANI can also promote cell differentiation. In order to deeply characterize the modifications of the bio-functionality induced by the cell-substrate interaction, the functional properties of the cells have been characterized by electrophysiology and Raman spectroscopy. Our results confirm that the PANI films do not strongly affect the general properties of the cells, ensuring their viability without toxic effects on their physiology. Ascribed to the adhesion process, however, a slight increase of the markers of the cell suffering has been evidenced by Raman spectroscopy and accordingly the electrophysiology shows a reduction at positive stimulations in the cells excitability.

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

Matrigel immobilization on the shish-kebab structured poly(ɛ-caprolactone) nanofibers for skin tissue engineering

NASA Astrophysics Data System (ADS)

Jing, Xin; Mi, Hao-Yang; Peng, Xiang-Fang; Turng, Lih-Sheng

2016-03-01

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ɛ-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface features of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.

Hyaluronic acid doped-poly(3,4-ethylenedioxythiophene)/chitosan/gelatin (PEDOT-HA/Cs/Gel) porous conductive scaffold for nerve regeneration.

PubMed

Wang, Shuping; Guan, Shui; Zhu, Zhibo; Li, Wenfang; Liu, Tianqing; Ma, Xuehu

2017-02-01

Conducting polymer, as a "smart" biomaterial, has been increasingly used to construct tissue engineered scaffold for nerve tissue regeneration. In this study, a novel porous conductive scaffold was prepared by incorporating conductive hyaluronic acid (HA) doped-poly(3,4-ethylenedioxythiophene) (PEDOT-HA) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physicochemical characteristics of Cs/Gel scaffold with 0-10wt% PEDOT-HA were analyzed and the results indicated that the incorporation of PEDOT-HA into scaffold increased the electrical and mechanical properties while decreasing the porosity and water absorption. Moreover, in vitro biodegradation of scaffold displayed a declining trend with the PEDOT-HA content increased. About the biocompatibility of conductive scaffold, neuron-like rat phaeochromocytoma (PC12) cells were cultured in scaffold to evaluate cell adhesion and growth. 8% PEDOT-HA/Cs/Gel scaffold had a higher cell adhesive efficiency and cell viability than the other conductive scaffolds. Furthermore, cells in the scaffold with 8wt% PEDOT-HA expressed higher synapse growth gene of GAP43 and SYP compared with Cs/Gel control group. These results suggest that 8%PEDOT-HA/Cs/Gel scaffold is an attractive cell culture conductive substrate which could support cell adhesion, survival, proliferation, and synapse growth for the application in nerve tissue regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

The role of surface implant treatments on the biological behavior of SaOS-2 osteoblast-like cells. An in vitro comparative study.

PubMed

Conserva, Enrico; Menini, Maria; Ravera, Giambattista; Pera, Paolo

2013-08-01

The aim of this study was an in vitro comparison of osteoblast adhesion, proliferation and differentiation related to six dental implants with different surface characteristics, and to determine if the interaction between cells and implant is influenced by surface structure and chemical composition. Six types of implants were tested, presenting four different surface treatments: turned, sandblasted, acid-etched, anodized. The implant macro- and microstructure were analyzed using SEM, and the surface chemical composition was investigated using energy-dispersive X-ray analysis. SaOS-2 osteoblasts were used for the evaluation of cell adhesion and proliferation by SEM, and cell viability in contact with the various surfaces was determined using cytotoxicity MTT assays. Alkaline phosphatase (ALP) enzymatic activity in contact with the six surfaces was evaluated. Data relative to MTT assay and ALP activity were statistically analyzed using Kruskal-Wallis not parametric test and Nemenyi-Damico-Wolfe-Dunn post hoc test. All the implants tested supported cell adhesion, proliferation and differentiation, revealing neither organic contaminants nor cytotoxicity effects. The industrial treatments investigated changed the implant surface microscopic aspect and SaOS-2 cell morphology appeared to be influenced by the type of surface treatment at 6, 24, and 72 h of growth. SaOS-2 cells spread more rapidly on sandblasted surfaces. Turned surfaces showed the lowest cell proliferation at SEM observation. Sandblasted surfaces showed the greatest ALP activity values per cell, followed by turned surfaces (P < 0.05). On the base of this in vitro investigation, differently surfaced implants affected osteoblast morphology, adhesion, proliferation, and differentiation. Sandblasted surfaces promoted the most suitable osteoblast behavior. © 2012 John Wiley & Sons A/S.

In vitro testing of commercial and potential probiotic lactic acid bacteria.

PubMed

Jensen, Hanne; Grimmer, Stine; Naterstad, Kristine; Axelsson, Lars

2012-02-01

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated. Copyright © 2011 Elsevier B.V. All rights reserved.

Study of the viability and adhesion of osteoblast cells to bone cements mixed with hydroxyapatite at different concentrations to use in vertebral augmentation techniques.

PubMed

Pino-Mínguez, J; Jorge-Mora, A; Couceiro-Otero, R; García-Santiago, C

2015-01-01

The purpose of this study is to compare the biocompatibility and the effect in osteoblasts of polymethyl methacrylate alone, and mixed with hydroxyapatite in different concentrations of 5, 10, 15 and 20%, without exceeding 20%, as it can alter mechanical properties of the composite. Experimental study comparing osteoblast response to Polymethyl methacrylate alone and with hydroxyapatite in different concentrations. Composites at 15 and 20% obtained better osteoblast response, with higher osteoblastic activity markers, and lower apoptosis markers. Electron microscopy images show improved adhesion of osteoblasts. Copyright © 2014 SECOT. Published by Elsevier Espana. All rights reserved.

Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae with host cells

PubMed Central

2010-01-01

Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. Results Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. Conclusions The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains. PMID:20942914

Investigation of Tumor Cell Behaviors on a Vascular Microenvironment-Mimicking Microfluidic Chip

PubMed Central

Huang, Rong; Zheng, Wenfu; Liu, Wenwen; Zhang, Wei; Long, Yunze; Jiang, Xingyu

2015-01-01

The extravasation of tumor cells is a key event in tumor metastasis. However, the mechanism underlying tumor cell extravasation remains unknown, mainly hindered by obstacles from the lack of complexity of biological tissues in conventional cell culture, and the costliness and ethical issues of in vivo experiments. Thus, a cheap, time and labor saving, and most of all, vascular microenvironment-mimicking research model is desirable. Herein, we report a microfluidic chip-based tumor extravasation research model which is capable of simultaneously simulating both mechanical and biochemical microenvironments of human vascular systems and analyzing their synergistic effects on the tumor extravasation. Under different mechanical conditions of the vascular system, the tumor cells (HeLa cells) had the highest viability and adhesion activity in the microenvironment of the capillary. The integrity of endothelial cells (ECs) monolayer was destroyed by tumor necrosis factor-α (TNF-α) in a hemodynamic background, which facilitated the tumor cell adhesion, this situation was recovered by the administration of platinum nanoparticles (Pt-NPs). This model bridges the gap between cell culture and animal experiments and is a promising platform for studying tumor behaviors in the vascular system. PMID:26631692

Laser-assisted nanoceramics reinforced polymer scaffolds for tissue engineering: additional heating and stem cells behavior

NASA Astrophysics Data System (ADS)

Shishkovsky, Igor; Scherbakov, Vladimir; Volchkov, Vladislav; Volova, Larisa

2018-02-01

The conditions of selective laser melting (SLM) of tissue engineering scaffolds affect cell response and must be engineered to support cell adhesion, proliferation, and differentiation. In the present study, the influence of additional heating during SLM process on stem cell viability near biopolymer matrix reinforced by nanoceramics additives was carried out. We used the biocompatible and bioresorbable polymers (polyetheretherketone /PEEK/ and polycaprolactone /PCL/) as a matrix and nano-oxide ceramics - TiO2, Al2O3, ZrO2, FexOy and/or hydroxyapatite as a basis of the additives. The rate of pure PEEK and PCL bio-resorption and in mixtures with nano oxides on the matrix was studied by the method of mass loss on bacteria of hydroxylase and enzyme complex. The stem cellular morphology, proliferative MMSC activity, and adhesion of the 2D and 3D nanocomposite matrices were the subjects of comparison. Medical potential of the SLS/M-fabricated nano-oxide ceramics after additional heating as the basis for tissue engineering scaffolds and cell targeting systems were discussed.

Biocompatibility enhancement of rare earth magnesium alloy by laser surface processing

NASA Astrophysics Data System (ADS)

Nie, Shilin; Wang, Yuqing; Liu, Haifeng; Guan, Yingchun

2018-01-01

Although magnesium and magnesium alloys are considered biocompatible and biodegradable, insufficient biocompatibility in body fluid environment is still the major drawback of magnesium alloys for their successful applications as biodegradable orthopaedic implants. In this work, magnesium alloy surface with both enhanced corrosion resistance and better cell adhesion property was directly fabricated by laser surface processing. Laser surface melting was used to improve corrosion resistance of Mg-6Gd-0.6Ca alloy. After laser surface melting, laser surface texturing was utilized on melted surface for better cell adhesion property. The corrosion resistance of laser-treated and as-received samples were evaluated using electrochemical technique. The effect of laser surface treatment on phase and microstructure evolution was evaluated using scanning electron microscopy, optical microscopy and X-ray diffraction. This work investigated the effect of laser treatment on cell distribution across the surface of magnesium alloy substrates. Osteoblast was cultured on the laser-treated surface and as-received surface. Cell morphology was observed with a scanning electron microscopy, and cell viability was evaluated by optical density measurement.

The effect of tributyltin on human eosinophilic [correction of eosinophylic] leukemia EoL-1 cells.

PubMed

Sroka, Jolanta; Włosiak, Przemysław; Wilk, Anna; Antonik, Justyna; Czyz, Jarosław; Madeja, Zbigniew

2008-01-01

Organotin compounds are chemicals that are widely used in industry and agriculture as plastic stabilizers, catalysts and biocides. Many of them, including tributyltin (TBT), have been detected in human food and, as a consequence, detectable levels have been found in human blood. As organotin compounds were shown to possess immunotoxic activity, we focused our attention on the effect of TBT on the basic determinants of the function of eosinophils, i.e. cell adhesiveness and motility. We used human eosinophylic leukemia EoL-1 cells, a common in vitro cellular model of human eosinophils. Here, we demonstrate that TBT causes a dose-dependent decrease in the viability of EoL-1 cells. When administered at sub-lethal concentrations, TBT significantly decreases the adhesion of EoL-1 cells to human fibroblasts (HSFs) and inhibits their migration on fibroblast surfaces. Since the basic function of eosinophils is to invade inflamed tissues, our results indicate that TBT, and possibly other organotin compounds, may affect major cellular properties involved in the determination of in vivo eosinophil function.

LL37 and hBD-3 elevate the β-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic.

PubMed

Chang, Hao-Teng; Tsai, Pei-Wen; Huang, Hsin-Hui; Liu, Yu-Shu; Chien, Tzu-Shan; Lan, Chung-Yu

2012-02-01

The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidaemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the AMPs (antimicrobial peptides) LL37 and hBD-3 (human β-defensin-3) inhibited C. albicans viability and its adhesion to plastic. For the present study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall β-1,3-exoglucanase Xog1p, which is involved in cell-wall β-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the decrease in adhesion. For the present study, Xog1p(41-438)-6H, an N-terminally truncated, active, recombinant construct of Xog1p and Xog1p fragments were produced and used in pull-down assays and ELISA in vitro, which demonstrated that all constructs interacted with both AMPs. Enzymatic analyses showed that LL37 and hBD-3 enhanced the β-1,3-exoglucanase activity of Xog1p(41-438)-6H approximately 2-fold. Therefore elevated Xog1p activity might compromise cell-wall integrity and decrease C. albicans adhesion. To test this hypothesis, C. albicans was treated with 1.3 μM Xog1p(41-438)-6H and C. albicans adhesion to plastic decreased 47.7%. Taken together, the evidence suggests that Xog1p is one of the LL37/hBD-3 targets, and elevated β-1,3-exoglucanase activity reduces C. albicans adhesion to plastic.

Effects of Magnolol and Honokiol on Adhesion, Yeast-Hyphal Transition, and Formation of Biofilm by Candida albicans

PubMed Central

Sun, Lingmei; Liao, Kai; Wang, Dayong

2015-01-01

Background The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary. Methods The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol. Results and conclusions Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans. General Significance This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection. PMID:25710475

Design of Xylose-Based Semisynthetic Polyurethane Tissue Adhesives with Enhanced Bioactivity Properties.

PubMed

Balcioglu, Sevgi; Parlakpinar, Hakan; Vardi, Nigar; Denkbas, Emir Baki; Karaaslan, Merve Goksin; Gulgen, Selam; Taslidere, Elif; Koytepe, Suleyman; Ates, Burhan

2016-02-01

Developing biocompatible tissue adhesives with high adhesion properties is a highly desired goal of the tissue engineering due to adverse effects of the sutures. Therefore, our work involves synthesis, characterization, adhesion properties, protein adsorption, in vitro biodegradation, in vitro and in vivo biocompatibility properties of xylose-based semisynthetic polyurethane (NPU-PEG-X) bioadhesives. Xylose-based semisynthetic polyurethanes were developed by the reaction among 4,4'-methylenebis(cyclohexyl isocyanate) (MCI), xylose and polyethylene glycol 200 (PEG). Synthesized polyurethanes (PUs) showed good thermal stability and high adhesion strength. The highest values in adhesion strength were measured as 415.0 ± 48.8 and 94.0 ± 2.8 kPa for aluminum substrate and muscle tissue in 15% xylose containing PUs (NPU-PEG-X-15%), respectively. The biodegradation of NPU-PEG-X-15% was also determined as 19.96 ± 1.04% after 8 weeks of incubation. Relative cell viability of xylose containing PU was above 86%. Moreover, 10% xylose containing NPU-PEG-X (NPU-PEG-X-10%) sample has favorable tissue response, and inflammatory reaction between 1 and 6 weeks implantation period. With high adhesiveness and biocompatibility properties, NPU-PEG-X can be used in the medical field as supporting materials for preventing the fluid leakage after abdominal surgery or wound closure.

An investigation of cell growth and detachment from thermoresponsive physically crosslinked networks.

PubMed

Healy, Deirdre; Nash, Maria; Gorleov, Alexander; Thompson, Kerry; Dockery, Peter; Rochev, Yury

2017-11-01

The primary aim of this investigation was to determine the biocompatibility and cell culture potential of a newly designed class of thermoresponsive polymers. The attractiveness of these polymers lies in the fact that they swell rather than dissolve when the temperature is reduced below their respective lower critical solution temperature, due to the incorporation of octadecyl methacrylate (ODMA). The ODMA monomer acts as a physical crosslinker, preventing polymer dissolution upon temperature reduction. Two polymers were studied in this investigation poly(N isorpoylacrylamide (NIPAm)(99.25%)-co-ODMA(0.75%)) and poly(NIPAm(65%)-co-N-tert-butylacrylamide (NtBAm)(34.25%)-co-ODMA(0.75%)). Thin thermoresponsive films of the polymers were prepared via spin coating. 3T3 cells were then seeded on the prepared films and cell viability was assessed quantitatively through cell viability and activity assays and qualitatively by light microscopy. Cells were successfully seeded and grown on the poly(NIPAm-co-ODMA) and poly(NIPAm-co-NtBAm-co-ODMA) copolymer films after film modification with cell adhesion promoters (CAPs). Cell sheets successfully detached from the CAP coated poly(NIPAm-co-ODMA) platforms upon temperature reduction. Copyright © 2017 Elsevier B.V. All rights reserved.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Apigenin Attenuates Melanoma Cell Migration by Inducing Anoikis through Integrin and Focal Adhesion Kinase Inhibition.

PubMed

Hasnat, Md Abul; Pervin, Mehnaz; Lim, Ji Hong; Lim, Beong Ou

2015-11-27

Apigenin, a nonmutagenic flavonoid, has been found to have antitumor properties and is therefore particularly relevant for the development of chemotherapeutic agents for cancers. In this study, time- and dose-dependent cell viability and cytotoxicity were assessed to determine the effects of apigenin on A2058 and A375 melanoma cells. Melanoma cells were pretreated with different concentrations of apigenin and analyzed for morphological changes, anoikis induction, cell migration, and levels of proteins associated with apoptosis. Apigenin reduced integrin protein levels and inhibited the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK1/2), which induce anoikis in human cutaneous melanoma cells. Apigenin exhibited dose-dependent inhibition of melanoma cell migration, unlike untreated controls. Furthermore, apigenin treatment increased apoptotic factors such as caspase-3 and cleaved poly(ADP-ribose) polymerase in a dose-dependent manner, demonstrating the metastasis of melanoma cells. Our results provide a new insight into the mechanisms by which apigenin prevents melanoma metastasis by sensitizing anoikis induced by the loss of integrin proteins in the FAK/ERK1/2 signaling pathway. These findings elucidate the related mechanisms and suggest the potential of apigenin in developing clinical treatment strategies against malignant melanoma.

Investigation of viability of plant tissue in the environmental scanning electron microscopy.

PubMed

Zheng, Tao; Waldron, K W; Donald, Athene M

2009-11-01

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.

Synthesis and Characterization of Carboxymethylcellulose-Methacrylate Hydrogel Cell Scaffolds

PubMed Central

Reeves, Robert; Ribeiro, Andreia; Lombardo, Leonard; Boyer, Richard; Leach, Jennie B.

2012-01-01

Many carbohydrates pose advantages for tissue engineering applications due to their hydrophilicity, degradability, and availability of chemical groups for modification. For example, carboxymethylcellulose (CMC) is a water-soluble cellulose derivative that is degradable by cellulase. Though this enzyme is not synthesized by mammalian cells, cellulase and the fragments derived from CMC degradation are biocompatible. With this in mind, we created biocompatible, selectively degradable CMC-based hydrogels that are stable in routine culture, but degrade when exposed to exogenous cellulase. Solutions of CMC-methacrylate and polyethylene glycol dimethacrylate (PEG-DM) were co-crosslinked to form stable hydrogels; we found that greater CMC-methacrylate content resulted in increased gel swelling, protein diffusion and rates of degradation by cellulase, as well as decreased gel shear modulus. CMC-methacrylate/PEG-DM gels modified with the adhesive peptide RGD supported fibroblast adhesion and viability. We conclude that hydrogels based on CMC-methacrylate are suitable for bioengineering applications where selective degradability may be favorable, such as cell scaffolds or controlled release devices. PMID:22708058

In Vitro Assessment of Early Bacterial Activity on Micro/Nanostructured Ti6Al4V Surfaces.

PubMed

Valdez-Salas, Benjamin; Beltrán-Partida, Ernesto; Castillo-Uribe, Sandra; Curiel-Álvarez, Mario; Zlatev, Roumen; Stoytcheva, Margarita; Montero-Alpírez, Gisela; Vargas-Osuna, Lidia

2017-05-18

It is imperative to understand and systematically compare the initial interactions between bacteria genre and surface properties. Thus, we fabricated a flat, anodized with 80 nm TiO₂ nanotubes (NTs), and a rough Ti6Al4V surface. The materials were characterized using field-emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM). We cultured in vitro Staphylococcus epidermidis ( S. epidermidis ) and Pseudomonas aeruginosa ( P. aeruginosa ) to evaluate the bacterial-surface behavior by FE-SEM and viability calculation. In addition, the initial effects of human osteoblasts were tested on the materials. Gram-negative bacteria showed promoted adherence and viability over the flat and rough surface, while NTs displayed opposite activity with altered morphology. Gram-positive bacteria illustrated similar cellular architecture over the surfaces but with promoted surface adhesion bonds on the flat alloy. Rough surfaces supported S. epidermidis viability, whilst NTs exhibited lower vitality. NTs advocated promoted better osteoblast organization with enhanced vitality. Gram-positive bacteria suggested preferred adhesion capability over flat and carbon-rich surfaces. Gram-negative bacteria were strongly disturbed by NTs but largely stimulated by flat and rough materials. Our work proposed that the chemical profile of the material surface and the bacterial cell wall characteristics might play an important role in the bacteria-surface interactions.

Synergistic effect of polyaniline coverage and surface microstructure on the inhibition of Pseudomonas aeruginosa biofilm formation.

PubMed

Gallarato, L A; Mulko, L E; Dardanelli, M S; Barbero, C A; Acevedo, D F; Yslas, E I

2017-02-01

Biofilm Formation is a survival strategy for microorganisms to adapt to their environment. Microbial cells in biofilm become tolerant and resistant to antibiotics and immune responses, increasing the difficulties for the clinical treatment of microbial infections. The surface chemistry and the micro/nano-topography of solid interfaces play a major role in mediating microorganism activity and adhesion. The effect of the surface chemical composition and topography on the adhesion and viability of Pseudomonas aeruginosa was studied. Polymeric (polyethylene terephthalate) surfaces were covered with a conducting polymer (polyaniline, PANI) film by in-situ polymerization and microstructured by Direct Laser Interference Patterning (DLIP). The viability of Pseudomonas aeruginosa on the different surfaces was investigated. The physicochemical properties of the surfaces were characterized by water contact angle measurements, scanning electron microscopy and atomic force microscopy. Bacterial biofilms were imaged by atomic force and scanning electron microscopies. The bacterial viability decreased on PANI compared with the substrate (polyethylene terephthalate) and it decreased even more upon micro-structuring the PANI films. In addition, the biofilm reduction could be improved using polymers with different chemical composition and/or the same polymer with different topographies. Both methods presented diminish the bacterial attachment and biofilm formation. These findings present a high impact related to materials for biomedical engineer applications regarding medical devices, as prostheses or catheters. Copyright © 2016 Elsevier B.V. All rights reserved.

Dexamethasone and azathioprine promote cytoskeletal changes and affect mesenchymal stem cell migratory behavior.

PubMed

Schneider, Natália; Gonçalves, Fabiany da Costa; Pinto, Fernanda Otesbelgue; Lopez, Patrícia Luciana da Costa; Araújo, Anelise Bergmann; Pfaffenseller, Bianca; Passos, Eduardo Pandolfi; Cirne-Lima, Elizabeth Obino; Meurer, Luíse; Lamers, Marcelo Lazzaron; Paz, Ana Helena

2015-01-01

Glucocorticoids and immunosuppressive drugs are commonly used to treat inflammatory disorders, such as inflammatory bowel disease (IBD), and despite a few improvements, the remission of IBD is still difficult to maintain. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have emerged as regulators of the immune response, and their viability and activation of their migratory properties are essential for successful cell therapy. However, little is known about the effects of immunosuppressant drugs used in IBD treatment on MSC behavior. The aim of this study was to evaluate MSC viability, nuclear morphometry, cell polarity, F-actin and focal adhesion kinase (FAK) distribution, and cell migratory properties in the presence of the immunosuppressive drugs azathioprine (AZA) and dexamethasone (DEX). After an initial characterization, MSCs were treated with DEX (10 μM) or AZA (1 μM) for 24 hrs or 7 days. Neither drug had an effect on cell viability or nuclear morphometry. However, AZA treatment induced a more elongated cell shape, while DEX was associated with a more rounded cell shape (P < 0.05) with a higher presence of ventral actin stress fibers (P < 0.05) and a decrease in protrusion stability. After 7 days of treatment, AZA improved the cell spatial trajectory (ST) and increased the migration speed (24.35%, P < 0.05, n = 4), while DEX impaired ST and migration speed after 24 hrs and 7 days of treatment (-28.69% and -25.37%, respectively; P < 0.05, n = 4). In conclusion, our data suggest that these immunosuppressive drugs each affect MSC morphology and migratory capacity differently, possibly impacting the success of cell therapy.

Basic characteristics of Sporolactobacillus inulinus BCRC 14647 for potential probiotic properties.

PubMed

Huang, Hui-Ying; Huang, Shih-Yi; Chen, Pei-Yu; King, V An-Erl; Lin, Yeu-Pyng; Tsen, Jen-Horng

2007-05-01

The basic characteristics of the spore-forming lactic acid bacterium, Sporolactobacillus inulinus BCRC 14647, was evaluated in vitro for its potential probiotic properties. Assessments including acid and bile salt tolerance, adhesiveness, and antagonistic effects on pathogenic Salmonella enteritidis BCRC 10744, as well as inhibition factors of spent culture supernatant (SCS) and an invasion assay, were conducted using Lactobacillus acidophilus BCRC 10695 and two bifidobacteria (Bifidobacterium bifidum BCRC 14615 and B. longum BCRC 11847) as a reference. In the results, S. inulinus spores presented significantly higher survival rates than the vegetative cell form in acidic conditions as well as the reference bifidobacteria. However, L. acidophilus showed the highest viability among all tested strains. Similar results were found in the bile tolerance test. Compared with the reference strains, the vegetative cell form of S. inulinus possessed a proper adhesive characteristic (71.7 bacteria/field for S. inulinus and 91.3 and 45.7 bacteria/field for B. bifidum and B. longum, respectively). In the adhesion assay, both the spore form of S. inulinus (17.1 bacteria/field) and the negative control, L. bulgaricus BCRC 14009 (5.9 bacteria/field), displayed nonadhesive traits. The vegetative cells of S. inulinus and its SCS both dramatically decrease the adhesion of S. enteritidis to Caco-2 cells; meanwhile, the SCS of S. inulinus vegetative cells inhibited the growth of S. enteritidis in the inhibition zone test. The existing inhibition factor could be assumed to be lactic acid in the SCS. From the results of the invasion assay, S. inulinus showed high safety properties. In conclusion, based on these in vitro evaluations, results suggest that S. inulinus presents probiotic features of great potential in the vegetative cell form.

Highly cytocompatible and flexible three-dimensional graphene/polydimethylsiloxane composite for culture and electrochemical detection of L929 fibroblast cells.

PubMed

Waiwijit, Uraiwan; Maturos, Thitima; Pakapongpan, Saithip; Phokharatkul, Ditsayut; Wisitsoraat, Anurat; Tuantranont, Adisorn

2016-08-01

Recently, three-dimensional graphene interconnected network has attracted great interest as a scaffold structure for tissue engineering due to its high biocompatibility, high electrical conductivity, high specific surface area and high porosity. However, free-standing three-dimensional graphene exhibits poor flexibility and stability due to ease of disintegration during processing. In this work, three-dimensional graphene is composited with polydimethylsiloxane to improve the structural flexibility and stability by a new simple two-step process comprising dip coating of polydimethylsiloxane on chemical vapor deposited graphene/Ni foam and wet etching of nickel foam. Structural characterizations confirmed an interconnected three-dimensional multi-layer graphene structure with thin polydimethylsiloxane scaffold. The composite was employed as a substrate for culture of L929 fibroblast cells and its cytocompatibility was evaluated by cell viability (Alamar blue assay), reactive oxygen species production and vinculin immunofluorescence imaging. The result revealed that cell viability on three-dimensional graphene/polydimethylsiloxane composite increased with increasing culture time and was slightly different from a polystyrene substrate (control). Moreover, cells cultured on three-dimensional graphene/polydimethylsiloxane composite generated less ROS than the control at culture times of 3-6 h. The results of immunofluorescence staining demonstrated that fibroblast cells expressed adhesion protein (vinculin) and adhered well on three-dimensional graphene/polydimethylsiloxane surface. Good cell adhesion could be attributed to suitable surface properties of three-dimensional graphene/polydimethylsiloxane with moderate contact angle and small negative zeta potential in culture solution. The results of electrochemical study by cyclic voltammetry showed that an oxidation current signal with no apparent peak was induced by fibroblast cells and the oxidation current at an oxidation potential of +0.9 V increased linearly with increasing cell number. Therefore, the three-dimensional graphene/polydimethylsiloxane composite exhibits high cytocompatibility and can potentially be used as a conductive substrate for cell-based electrochemical sensing. © The Author(s) 2016.

Effects of amine fluoride on biofilm growth and salivary pellicles.

PubMed

van der Mei, H C; Engels, E; de Vries, J; Busscher, H J

2008-01-01

The amine fluoride (AmF) N'-octadecyl-trimethylene-diamine-N,N,N'-tris(2-ethanol)-dihydro-fluoride is a cationic antimicrobial which can have beneficial effects on plaque formation. Here, we determine changes in pellicle and bacterial cell surface properties of the strains Actinomyces naeslundii HM1, Streptococcus mutans NS, S.mutans ATCC 700610, S. sobrinus HG1025 and S. oralis HM1 upon adsorption of this AmF and accompanying effects on bacterial adhesion and biofilm growth. In vitro pellicles had a zeta potential of -12 mV that became less negative upon adsorption of AmF. The chemical functionalities in which carbon and oxygen were involved changed after AmF adsorption and AmF-treated pellicles had a greater surface roughness than untreated pellicles. Water contact angles in vitro decreased from 56 to 45 degrees upon AmF treatment, which corresponded with water contact angles (44 degrees ) measured intraorally on the front incisors of volunteers immediately after using an AmF-containing toothpaste. All bacterial strains were negatively charged and their isoelectric points (IEP) increased upon AmF adsorption. Minimal inhibitory concentrations were smallest for strains exhibiting the largest increase in IEP. Adhesion to salivary pellicles and biofilm growth of the mutans streptococcal strains were significantly reduced after AmF treatment, but not of A. naeslundii or S. oralis. However, regardless of the strain involved, biofilm viability decreased significantly after AmF treatment. The electrostatic interaction between cationic AmF and negatively charged bacterial cell surfaces is pivotal in establishing reduced biofilm formation by AmF through a combination of effects on initial adhesion and killing. The major effect of AmF treatment, however, was a reduction brought about in biofilm viability.

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth.

PubMed

Wang, Dong-an; Ji, Jian; Sun, Yong-hong; Shen, Jia-cong; Feng, Lin-xian; Elisseeff, Jennifer H

2002-01-01

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Using self-assembled monolayers to pattern ECM proteins and cells on substrates.

PubMed

Ostuni, Emanuele; Whitesides, George M; Ingber, Donald E; Chen, Christopher S

2009-01-01

We present a method that uses microcontact printing of alkanethiols on gold to generate patterned substrates presenting "islands" of extracellular matrix (ECM) surrounded by nonadhesive regions such that single cells attach and spread only on the adhesive regions. We have used this micropatterning technology to demonstrate that mammalian cells can be switched between growth and apoptosis programs in the presence of saturating concentrations of growth factors by either promoting or preventing cell spreading (Science 276:1425-1428, 1997). From the perspective of fundamental cell biology, these results suggested that the local differentials in growth and viability that are critical for the formation of complex tissue patterns may be generated by local changes in cell-ECM interactions. In the context of cell culture technologies, such as bioreactors and cellular engineering applications, the regulation of cell function by cell shape indicates that the adhesive microenvironment around cells can be carefully optimized by patterning a substrate in addition to using soluble factors (Biotech. Prog. 14:356-363, 1998). Micropatterning technology is playing a central role both in our understanding how ECM and cell shape regulate cell physiology and in facilitating the development of cellular biosensor and tissue engineering applications (Science 264:696-698, 1994; J. Neurosci. Res. 13:213-20, 1985; Biotech. Bioeng. 43:792-800, 1994).

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts.

PubMed

Vicente, Carolina Meloni; Ricci, Ritchelli; Nader, Helena Bonciani; Toma, Leny

2013-05-25

The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

JAM-A as a prognostic factor and new therapeutic target in multiple myeloma.

PubMed

Solimando, A G; Brandl, A; Mattenheimer, K; Graf, C; Ritz, M; Ruckdeschel, A; Stühmer, T; Mokhtari, Z; Rudelius, M; Dotterweich, J; Bittrich, M; Desantis, V; Ebert, R; Trerotoli, P; Frassanito, M A; Rosenwald, A; Vacca, A; Einsele, H; Jakob, F; Beilhack, A

2018-03-01

Cell adhesion in the multiple myeloma (MM) microenvironment has been recognized as a major mechanism of MM cell survival and the development of drug resistance. Here we addressed the hypothesis that the protein junctional adhesion molecule-A (JAM-A) may represent a novel target and a clinical biomarker in MM. We evaluated JAM-A expression in MM cell lines and in 147 MM patient bone marrow aspirates and biopsies at different disease stages. Elevated JAM-A levels in patient-derived plasma cells were correlated with poor prognosis. Moreover, circulating soluble JAM-A (sJAM-A) levels were significantly increased in MM patients as compared with controls. Notably, in vitro JAM-A inhibition impaired MM migration, colony formation, chemotaxis, proliferation and viability. In vivo treatment with an anti-JAM-A monoclonal antibody (αJAM-A moAb) impaired tumor progression in a murine xenograft MM model. These results demonstrate that therapeutic targeting of JAM-A has the potential to prevent MM progression, and lead us to propose JAM-A as a biomarker in MM, and sJAM-A as a serum-based marker for clinical stratification.

Development of VEGF-loaded PLGA matrices in association with mesenchymal stem cells for tissue engineering

PubMed Central

Rosa, A.R.; Steffens, D.; Santi, B.; Quintiliano, K.; Steffen, N.; Pilger, D.A.; Pranke, P.

2017-01-01

The association of bioactive molecules, such as vascular endothelial growth factor (VEGF), with nanofibers facilitates their controlled release, which could contribute to cellular migration and differentiation in tissue regeneration. In this research, the influence of their incorporation on a polylactic-co-glycolic acid (PLGA) scaffold produced by electrospinning on cell adhesion and viability and cytotoxicity was carried out in three groups: 1) PLGA/BSA/VEGF; 2) PLGA/BSA, and 3) PLGA. Morphology, fiber diameter, contact angle, loading efficiency and controlled release of VEGF of the biomaterials, among others, were measured. The nanofibers showed smooth surfaces without beads and with interconnected pores. PLGA/BSA/VEGF showed the smallest water contact angle and VEGF released for up to 160 h. An improvement in cell adhesion was observed for the PLGA/BSA/VEGF scaffolds compared to the other groups and the scaffolds were non-toxic for the cells. Therefore, the scaffolds were shown to be a good strategy for sustained delivery of VEGF and may be a useful tool for tissue engineering. PMID:28793048

Matrigel immobilization on the shish-kebab structured poly(ε-caprolactone) nanofibers for skin tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jing, Xin, E-mail: jingxinscut@gmail.com; Mi, Hao-Yang; Wisconsin Institutes for Discovery, University of Wisconsin-Madison, 53715

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ε-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface featuresmore » of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.« less

Carbon nanotube multilayered nanocomposites as multifunctional substrates for actuating neuronal differentiation and functions of neural stem cells.

PubMed

Shao, Han; Li, Tingting; Zhu, Rong; Xu, Xiaoting; Yu, Jiandong; Chen, Shengfeng; Song, Li; Ramakrishna, Seeram; Lei, Zhigang; Ruan, Yiwen; He, Liumin

2018-08-01

Carbon nanotubes (CNTs) have shown potential applications in neuroscience as growth substrates owing to their numerous unique properties. However, a key concern in the fabrication of homogeneous composites is the serious aggregation of CNTs during incorporation into the biomaterial matrix. Moreover, the regulation mechanism of CNT-based substrates on neural differentiation remains unclear. Here, a novel strategy was introduced for the construction of CNT nanocomposites via layer-by-layer assembly of negatively charged multi-walled CNTs and positively charged poly(dimethyldiallylammonium chloride). Results demonstrated that the CNT-multilayered nanocomposites provided a potent regulatory signal over neural stem cells (NSCs), including cell adhesion, viability, differentiation, neurite outgrowth, and electrophysiological maturation of NSC-derived neurons. Importantly, the dynamic molecular mechanisms in the NSC differentiation involved the integrin-mediated interactions between NSCs and CNT multilayers, thereby activating focal adhesion kinase, subsequently triggering downstream signaling events to regulate neuronal differentiation and synapse formation. This study provided insights for future applications of CNT-multilayered nanomaterials in neural fields as potent modulators of stem cell behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-10-01

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

Novel platform for minimizing cell loss on separation process: Droplet-based magnetically activated cell separator

NASA Astrophysics Data System (ADS)

Kim, Youngho; Hong, Su; Lee, Sang Ho; Lee, Kangsun; Yun, Seok; Kang, Yuri; Paek, Kyeong-Kap; Ju, Byeong-Kwon; Kim, Byungkyu

2007-07-01

To reduce the problem of cell loss due to adhesion, one of the basic phenomena in microchannel, we proposed the droplet-based magnetically activated cell separator (DMACS). Based on the platform of the DMACS—which consists of permanent magnets, a coverslip with a circle-shaped boundary, and an injection tube—we could collect magnetically (CD45)-labeled (positive) cells with high purity and minimize cell loss due to adhesion. To compare separation efficiency between the MACS and the DMACS, the total number of cells before and after separation with both the separators was counted by flow cytometry. We could find that the number (3241/59940) of cells lost in the DMACS is much less than that (22360/59940) in the MACS while the efficiency of cell separation in the DMACS (96.07%) is almost the same as that in the MACS (96.72%). Practically, with fluorescent images, it was visually confirmed that the statistical data are reliable. From the viability test by using Hoechst 33 342, it was also demonstrated that there was no cell damage on a gas-liquid interface. Conclusively, DMACS will be a powerful tool to separate rare cells and applicable as a separator, key component of lab-on-a-chip.

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

PubMed

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. Copyright © 2015. Published by Elsevier B.V.

Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

PubMed

Wiltshire, Rachael; Nelson, Vicky; Kho, Dan Ting; Angel, Catherine E; O'Carroll, Simon J; Graham, E Scott

2016-01-27

Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

The addition of inulin and Lactobacillus casei 01 in sheep milk ice cream.

PubMed

Balthazar, Celso F; Silva, Hugo L A; Esmerino, Erick A; Rocha, Ramon S; Moraes, Jeremias; Carmo, Mariana A V; Azevedo, Luciana; Camps, Ihosvany; K D Abud, Yuri; Sant'Anna, Celso; Franco, Robson M; Freitas, Mônica Q; Silva, Marcia C; Raices, Renata S L; Escher, Graziela B; Granato, Daniel; Senaka Ranadheera, C; Nazarro, Filomena; Cruz, Adriano G

2018-04-25

The effect of the Lactobacillus casei 01 and inulin addition on sheep milk ice cream during storage (-18 °C, 150 days) was investigated. Control, probiotic and synbiotic ice cream (10% w/w sheep milk cream; 10% w/w sheep milk cream, L. casei 01, 6 log CFU/mL; 10% w/w inulin, L. casei 01, 6 log CFU/mL, respectively) were manufactured. Microbiological counts (probiotic count, survival after in vitro gastrointestinal resistance, Caco-2 cell adhesion), bioactivity and microstructure were analysed. Physical and textural characteristics, colour parameters, thermal analysis and organic acids/volatile compounds were also evaluated. All formulations supported L. casei 01 viability and maintained above the minimum therapeutic level (>6 log CFU/mL) during storage. Inulin did not affect L. casei 01 survival after the passage through simulated gastrointestinal tract and adhesion to Caco-2 cells while improved the ACE-inhibitory and antioxidant activity. L. casei 01 addition produced several volatile compounds, such as carboxylic acids, alcohols, aldehydes and ketones. Also, scanning electron microscopy showed an interaction between probiotic bacteria and inulin fibre on synbiotic ice cream and the adhesion of L. casei to Caco-2 cells was observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platelet impedance adhesiometry: A novel technique for the measurement of platelet adhesion and spreading.

PubMed

Polgár, L; Soós, P; Lajkó, E; Láng, O; Merkely, B; Kőhidai, L

2018-06-01

Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function. © 2018 John Wiley & Sons Ltd.

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

PubMed

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

2013-05-01

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

Inhibition of infection and transmission of HIV-1 and lack of significant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents.

PubMed

Petrova, Mariya I; Mathys, Leen; Lebeer, Sarah; Noppen, Sam; Van Damme, Els J M; Tanaka, Haruo; Igarashi, Yasuhiro; Vaneechoutte, Mario; Vanderleyden, Jos; Balzarini, Jan

2013-09-01

A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV-1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.

Design of a novel magnetic platform for cell manipulation

NASA Astrophysics Data System (ADS)

Lucarini, Gioia; Iacovacci, Veronica; Gouveia, Pedro J.; Ricotti, Leonardo; Menciassi, Arianna

2018-02-01

Cell manipulation tasks, especially in lab-on-a-chip applications for personalized medicine, could greatly benefit from mobile untethered microdevices able to wirelessly navigate in fluidic environments by means of magnetic fields. In this paper, the design, fabrication and testing of a magnetic platform enabling the controlled locomotion and immersion of microrobots placed at the air/liquid interface is proposed and exploited for cell manipulation. The proposed microrobot consists of a polymeric magnetic thin film that acts as cell transporter and a specific coating strategy, devised to enhance a safe cancer cell adhesion to the magnetic film. Experimental results demonstrated an overall cell viability and a fine control of magnetic microrobot locomotion. The proposed technologies are promising in view of future cell manipulation tasks for personalized medicine applications.

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

PubMed Central

Brito, Jose L.R.; Walker, Brian; Jenner, Matthew; Dickens, Nicholas J.; Brown, Nicola J.M.; Ross, Fiona M.; Avramidou, Athanasia; Irving, Julie A.E.; Gonzalez, David; Davies, Faith E.; Morgan, Gareth J.

2009-01-01

Background The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Design and Methods The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. Results We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival. PMID:19059936

Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

PubMed

Angely, Christelle; Nguyen, Ngoc-Minh; Andre Dias, Sofia; Planus, Emmanuelle; Pelle, Gabriel; Louis, Bruno; Filoche, Marcel; Chenal, Alexandre; Ladant, Daniel; Isabey, Daniel

2017-08-01

The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure <1 h. At 30 min of exposure, CyaA toxin has a minimal effect on cell viability (>95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells. These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells

PubMed Central

2010-01-01

Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein. Results Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these. Conclusions Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface. PMID:20051108

Osteoblast response to porous titanium and biomimetic surface: In vitro analysis.

PubMed

Prado, Renata Falchete do; de Oliveira, Fernanda Saraiva; Nascimento, Rodrigo Dias; de Vasconcellos, Luana Marotta Reis; Carvalho, Yasmin Rodarte; Cairo, Carlos Alberto Alves

2015-01-01

This study analyzed the behavior of human osteoblasts cultured on porous titanium specimens, with and without biomimetic treatment, compared to dense titanium. The experiment had seven groups: Group 1: cells cultured on polystyrene of culture plate wells; Group 2: cells cultured on dense titanium specimen; Group 3: specimen with 33.79% of pores; Group 4: 41.79% of pores; Groups 5, 6 and 7: specimens similar to groups 2, 3 and 4, yet with biomimetic treatment. Real time-polymerase chain reaction with reverse transcription of the following genes was performed: prostaglandin E2 synthase, integrin β1, osterix, Runx2, Interleukin 6, macrophage colony stimulating factor, apolipoprotein E and others. The study achieved data on cell adhesion, growth and viability, total protein content, alkaline phosphatase activity and quantity of mineralized nodule formations. Data were statistically evaluated. Adherent cells and alkaline phosphatase activity were similar in titanium specimens, regardless of the groups. Biomimetic treatment reduced the total protein activity and the viability of tested cells. Most tested genes had statistically similar expression in all groups. The tested porosities did not cause alterations in osteoblast behavior and the biomimetic treatment impaired the biocompatibility of titanium causing cytotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Promising Ta-Ti-Zr-Si metallic glass coating without cytotoxic elements for bio-implant applications

NASA Astrophysics Data System (ADS)

Lai, J. J.; Lin, Y. S.; Chang, C. H.; Wei, T. Y.; Huang, J. C.; Liao, Z. X.; Lin, C. H.; Chen, C. H.

2018-01-01

Tantalum (Ta) is considered as one of the most promising metal due to its high corrosion resistance, excellent biocompatibility and cell adhesion/in-growth capabilities. Although there are some researches exploring the biomedical aspects of Ta and Ta based alloys, systematic characterizations of newly developed Ta-based metallic glasses in bio-implant applications is still lacking. This study employs sputtering approach to produced thin-film Ti-based metallic glasses due to the high melting temperature of Ta (3020 °C). Two fully amorphous Ta-based metallic glasses composed of Ta57Ti17Zr15Si11 and Ta75Ti10Zr8Si7 are produced and experimentally characterized in terms of their mechanical properties, bio-corrosion properties, surface hydrophilic characteristics, and in-vitro cell viability and cells attachment tests. Compare to conventional pure Ti and Ta metals, the developed Ta-based metallic glasses exhibit higher hardness and lower modulus which are better match to the mechanical properties of bone. MTS assay results show that Ta-based metallic glasses show comparable cell viability and cell attachment rate compared to that of pure Ti and Ta surface in a 72 h in-vitro test.

Comparison of cell behavior on pva/pva-gelatin electrospun nanofibers with random and aligned configuration

NASA Astrophysics Data System (ADS)

Huang, Chen-Yu; Hu, Keng-Hsiang; Wei, Zung-Hang

2016-12-01

Electrospinning technique is able to create nanofibers with specific orientation. Poly(vinyl alcohol) (PVA) have good mechanical stability but poor cell adhesion property due to the low affinity of protein. In this paper, extracellular matrix, gelatin is incorporated into PVA solution to form electrospun PVA-gelatin nanofibers membrane. Both randomly oriented and aligned nanofibers are used to investigate the topography-induced behavior of fibroblasts. Surface morphology of the fibers is studied by optical microscopy and scanning electron microscopy (SEM) coupled with image analysis. Functional group composition in PVA or PVA-gelatin is investigated by Fourier Transform Infrared (FTIR). The morphological changes, surface coverage, viability and proliferation of fibroblasts influenced by PVA and PVA-gelatin nanofibers with randomly orientated or aligned configuration are systematically compared. Fibroblasts growing on PVA-gelatin fibers show significantly larger projected areas as compared with those cultivated on PVA fibers which p-value is smaller than 0.005. Cells on PVA-gelatin aligned fibers stretch out extensively and their intracellular stress fiber pull nucleus to deform. Results suggest that instead of the anisotropic topology within the scaffold trigger the preferential orientation of cells, the adhesion of cell membrane to gelatin have substantial influence on cellular behavior.

A novel derivative of decursin, CSL-32, blocks migration and production of inflammatory mediators and modulates PI3K and NF-κB activities in HT1080 cells.

PubMed

Lee, Seung-Hee; Lee, Jee Hyun; Kim, Eun-Ju; Kim, Won-Jung; Suk, Kyoungho; Kim, Joo-Hwan; Song, Gyu Yong; Lee, Won-Ha

2012-07-01

Decursin and related coumarin compounds in herbal extracts have a number of biological activities against inflammation, angiogenesis and cancer. We have analysed a derivative of decursin (CSL-32) for activity against inflammatory activation of cancer cells, such as migration, invasion and expression of pro-inflammatory mediators. The human fibrosarcoma cell line, HT1080, was treated with TNFα (tumour necrosis factor α) in the presence or absence of CSL-32. The cellular responses and modification of signalling adapters were analysed with respect to the production of pro-inflammatory mediators, as also migration, adhesion and invasion. Treatment of HT1080 cells with CSL-32 inhibited their proliferation, without affecting cell viability, and TNFα-induced expression of pro-inflammatory mediators, such as MMP-9 (matrix metalloproteinase-9) and IL-8 (interleukin-8). CSL-32 also suppressed phosphorylation and degradation of IκB (inhibitory κB), phosphorylation of p65 subunit of NF-κB (nuclear factor-κB) and nuclear translocation of NF-κB, which are required for the expression of pro-inflammatory mediators. In addition, CSL-32 inhibited invasion and migration of HT1080 cells, as also cellular adhesion to fibronectin, an ECM (extracellular matrix) protein. CSL-32 treatment resulted in a dose-dependent inhibition of PI3K (phosphoinositide 3-kinase) activity, required for the cellular migration. The analyses show that CSL-32 inhibits processes associated with inflammation, such as the production of pro-inflammatory mediators, as well as adhesion, migration and invasion in HT1080 cells.

Surface Modification of Biodegradable Polymers towards Better Biocompatibility and Lower Thrombogenicity

PubMed Central

Rudolph, Andreas; Teske, Michael; Illner, Sabine; Kiefel, Volker; Sternberg, Katrin; Grabow, Niels; Wree, Andreas; Hovakimyan, Marina

2015-01-01

Purpose Drug-eluting stents (DES) based on permanent polymeric coating matrices have been introduced to overcome the in stent restenosis associated with bare metal stents (BMS). A further step was the development of DES with biodegradable polymeric coatings to address the risk of thrombosis associated with first-generation DES. In this study we evaluate the biocompatibility of biodegradable polymer materials for their potential use as coating matrices for DES or as materials for fully bioabsorbable vascular stents. Materials and Methods Five different polymers, poly(L-lactide) PLLA, poly(D,L-lactide) PDLLA, poly(L-lactide-co-glycolide) P(LLA-co-GA), poly(D,L-lactide-co-glycolide) P(DLLA-co-GA) and poly(L-lactide-co-ε-caprolactone), P(LLA-co-CL) were examined in vitro without and with surface modification. The surface modification of polymers was performed by means of wet-chemical (NaOH and ethylenediamine (EDA)) and plasma-chemical (O2 and NH3) processes. The biocompatibility studies were performed on three different cell types: immortalized mouse fibroblasts (cell line L929), human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). The biocompatibility was examined quantitatively using in vitro cytotoxicity assay. Cells were investigated immunocytochemically for expression of specific markers, and morphology was visualized using confocal laser scanning (CLSM) and scanning electron (SEM) microscopy. Additionally, polymer surfaces were examined for their thrombogenicity using an established hemocompatibility test. Results Both endothelial cell types exhibited poor viability and adhesion on all five unmodified polymer surfaces. The biocompatibility of the polymers could be influenced positively by surface modifications. In particular, a reproducible effect was observed for NH3-plasma treatment, which enhanced the cell viability, adhesion and morphology on all five polymeric surfaces. Conclusion Surface modification of polymers can provide a useful approach to enhance their biocompatibility. For clinical application, attempts should be made to stabilize the plasma modification and use it for coupling of biomolecules to accelerate the re-endothelialization of stent surfaces in vivo. PMID:26641662

Endocytic mechanisms and osteoinductive profile of hydroxyapatite nanoparticles in human umbilical cord Wharton’s jelly-derived mesenchymal stem cells

PubMed Central

Zhang, Juan; Wang, Chen

2018-01-01

Background As a potentially bioactive material, the widespread application of nanosized hydroxyapatite (nano-HAP) in the field of bone regeneration has increased the risk of human exposure. However, our understanding of the interaction between nano-HAP and stem cells implicated in bone repair remains incomplete. Methods Here, we characterized the adhesion and cellular internalization of HAP nanoparticles (HANPs) with different sizes (20 nm np20 and 80 nm np80) and highlighted the involved pathway in their uptake using human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs). In addition, the effects of HANPs on cell viability, apoptosis response, osteogenic differentiation, and underlying related mechanisms were explored. Results It was shown that both types of HANPs readily adhered to the cellular membrane and were transported into the cells compared to micro-sized HAP particles (m-HAP; 12 μm). Interestingly, the endocytic routes of np20 and np80 differed, although they exhibited similar kinetics of adhesion and uptake. Our study revealed involvement of clathrin- and caveolin-mediated endocytosis as well as macropinocytosis in the np20 uptake. However, for np80, clathrin-mediated endocytosis and some as-yet-unidentified important uptake routes play central roles in their internalization. HANPs displayed a higher preference to accumulate in the cytoplasm compared to m-HAP, and HANPs were not detected in the nucleolus. Exposure to np20 for 24 h caused a decrease in cell viability, while cells completely recovered with an exposure time of 72 h. Furthermore, HANPs did not influence apoptosis and necrosis of hWJ-MSCs. Strikingly, HANPs enhanced mRNA levels of osteoblast-related genes and stimulated calcium mineral deposition, and this directly correlated with the activation in c-Jun N-terminal kinases and p38 pathways. Conclusion Our data provide additional insight about the interactions of HANPs with MSCs and suggest their application potential in hard tissue regeneration. PMID:29559775

Assessment of Parylene C Thin Films for Heart Valve Tissue Engineering

PubMed Central

Marei, Isra; Chester, Adrian; Carubelli, Ivan; Prodromakis, Themistoklis; Trantidou, Tatiana

2015-01-01

Background: Scaffolds are a key component of tissue-engineered heart valves (TEHVs). Several approaches had been adopted in the design of scaffolds using both natural and synthetic resources. We have investigated the suitability of parylene C (PC), a vapor deposited polymeric material, for the use as a scaffold in TEHV. Aims: To evaluate the adsorption of extracellular matrix components onto plasma-activated PC and study the biocompatibility of PC by measuring cellular adhesion, viability, apoptosis, and phenotypic expression of valve endothelial and interstitial cells. Finally, the mechanical properties of PC were compared with those of native aortic valve cusp tissue. Methods: PC slides were plasma activated and then coated with gelatin, type I collagen, or fibronectin. Porcine pulmonary valve endothelial and interstitial cells were then grown on plasma oxidized PC with different types of coatings and their adhesion was observed after 20 h of incubation. Cell viability was tested using the MTS assay, and apoptosis was estimated using TUNEL staining. The mechanical properties of PC and valve tissue were measured using a Bose Mechanical Tester. Finally, cell-seeded PC films were exposed to pulsatile pressure and aortic shear stress, respectively, to test their durability in a dynamic environment. Results: Our findings show that collagen and fibronectin could bind to plasma oxidized PC. Both valve endothelial and interstitial cells adhered to protein-coated ECM. PC had a profile of mechanical stiffness and ultimate tensile strength that were comparable with or in excess of those seen in porcine aortic valve cusps. Cells were still attached to PC films after 3 days of exposure to up to 50 mmHg pulsatile pressure or aortic levels of shear stress. Conclusion: PC is a promising candidate for use as a scaffold in tissue engineering heart valves. Additional studies are required to determine both the durability and long-term performance of cell-seeded PC when in a similar hemodynamic environment to that of the aortic valve. PMID:26101808

Biological Analysis of Simvastatin-releasing Chitosan Scaffold as a Cell-free System for Pulp-dentin Regeneration.

PubMed

Soares, Diana G; Anovazzi, Giovanna; Bordini, Ester Alves F; Zuta, Uxua O; Silva Leite, Maria Luísa A; Basso, Fernanda G; Hebling, Josimeri; de Souza Costa, Carlos A

2018-06-01

The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system. First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 μmol/L (CHSC-SIM1.0) and 0.5 μmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro. SIM at 0.1 μmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional-cultured DPCs than on those in contact with CHSC. CHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs. Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Adhesion of CdTe quantum dots on model membranes and internalization into RBL-2H3 cells.

PubMed

Zhang, Mengmeng; Wei, Xiaoran; Ding, Lei; Hu, Jingtian; Jiang, Wei

2017-06-01

Quantum dots (QDs) have attracted broad attention due to their special optical properties and promising prospect in medical and biological applications. However, the process of QDs on cell membrane is worth further investigations because such process may lead to harmful effects on organisms and also important for QD application. In this study, adhesion of amino- and carboxyl-coated CdTe QDs (A-QDs and C-QDs) on cell membrane and the subsequent internalization are studied using a series of endocytosis-free model membranes, including giant and small unilamellar vesicles, supported lipid bilayers and giant plasma membrane vesicles (GPMVs). The adhered QD amounts on model membranes are quantified by a quartz crystal microbalance. The CdTe QD adhesion on model membranes is governed by electrostatic forces. Positively charged A-QDs adhere on GPMV surface and passively penetrate the plasma membrane via endocytosis-free mechanism, but negatively charged C-QDs cannot. Rat basophilic leukemia (RBL-2H3) cells are exposed to CdTe QDs to monitor the QD internalization process. Both A- and C-QDs are internalized by RBL-2H3 cells mainly via endocytosis. CdTe QDs do not accumulate on the plasma membrane of living cells due to the fast endocytosis and the weakened electrostatic attraction in biological medium, resulting in low chance of passive penetration. The suspended cells after trypsin digestion take more QDs than the adherent cells. A-QDs cause lower cell viability than C-QDs, probably because the approach of positively charged QDs to cells is favored and the smaller aggregates of A-QDs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of both focal adhesion kinase and fibroblast growth factor receptor 2 pathways induces anti-tumor and anti-angiogenic activities.

PubMed

Dao, Pascal; Jarray, Rafika; Smith, Nikaia; Lepelletier, Yves; Le Coq, Johanne; Lietha, Daniel; Hadj-Slimane, Réda; Herbeuval, Jean-Philippe; Garbay, Christiane; Raynaud, Françoise; Chen, Huixiong

2014-06-28

FAK and FGFR2 signaling pathways play important roles in cancer development, progression and tumor angiogenesis. PHM16 is a novel ATP competitive inhibitor of FAK and FGFR2. To evaluate the therapeutic efficacy of this agent, we examined its anti-angiogenic effect in HUVEC and its anti-tumor effect in different cancer cell lines. We showed PHM16 inhibited endothelial cell viability, adherence and tube formation along with the added ability to induce endothelial cell apoptosis. This compound significantly delayed tumor cell growth. Together, these data showed that inhibition of both FAK and FGFR2 signaling pathways can enhance anti-tumor and anti-angiogenic activities. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Influence of different air-abrasive powders on cell viability at biologically contaminated titanium dental implants surfaces.

PubMed

Schwarz, Frank; Ferrari, Daniel; Popovski, Kristian; Hartig, Brigitte; Becker, Jürgen

2009-01-01

Studies have indicated that oral biofilm formation at structured titanium surfaces interferes with cell adhesion and proliferation, and its removal by means of conventional treatment procedures may not be sufficient to render these surfaces biologically acceptable. Therefore, the aim of the study was to evaluate the influence of different air-abrasive powders on cell viability at biologically contaminated titanium dental implant surfaces. Intraoral splints were used to collect an in vivo biofilm on sandblasted and acid-etched titanium discs for 48 h. A single (1x) and repeated (2x) use of four different powders (amino acid glycine or sodium bicarbonate particles; range of mean particle size (d(v50)):20-75 microm) was applied at two distances (1 and 2 mm) and angles (30 degrees and 90 degrees) to the surfaces. Specimens (2x) were incubated with SaOs-2 cells for 7 days. Residual biofilm (RB) areas (%), and surface alterations (SEM) (1x and 2x), as well as SaOs-2 cell viability, expressed as mitochondrial cell activity (MA) (counts/second) (2x specimens), were assessed. Comparable mean RB areas were observed within and between groups after both 1x (RB: 0.0% +/- 0.0% to 5.7% +/- 5.7%) and 2x (RB: 0.0% +/- 0.0%) treatments. All surface treatments did not lead to MA (2x) values comparable to the sterile control group. However, sodium bicarbonate particles resulted in significantly higher MA (2x) values than amino acid glycine powders of different sizes. This was associated with pronounced alterations of the surface morphology (2x). Within the limits of the present study, it was concluded that SaOs-2 cell viability at biologically contaminated titanium surfaces was mainly influenced by the particle type of the powder. (c) 2008 Wiley Periodicals, Inc.

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells.

PubMed

Liu, Peng; Wang, Wei; Zhou, Zhigang; Smith, Andrew J O; Bowater, Richard P; Wormstone, Ian Michael; Chen, Yuqiong; Bao, Yongping

2018-05-09

Sulforaphane (SFN) exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane- N -acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs) and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H₂O₂ challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the induction of intracellular glutathione (GSH) played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

A new fibrin sealant as a three-dimensional scaffold candidate for mesenchymal stem cells

PubMed Central

2014-01-01

Introduction The optimization of an organic scaffold for specific types of applications and cells is vital to successful tissue engineering. In this study, we investigated the effects of a new fibrin sealant derived from snake venom as a scaffold for mesenchymal stem cells, to demonstrate the ability of cells to affect and detect the biological microenvironment. Methods The characterization of CD34, CD44 and CD90 expression on mesenchymal stem cells was performed by flow cytometry. In vitro growth and cell viability were evaluated by light and electron microscopy. Differentiation into osteogenic, adipogenic and chondrogenic lineages was induced. Results The fibrin sealant did not affect cell adhesion, proliferation or differentiation and allowed the adherence and growth of mesenchymal stem cells on its surface. Hoechst 33342 and propidium iodide staining demonstrated the viability of mesenchymal stem cells in contact with the fibrin sealant and the ability of the biomaterial to maintain cell survival. Conclusions The new fibrin sealant is a three-dimensional scaffolding candidate that is capable of maintaining cell survival without interfering with differentiation, and might also be useful in drug delivery. Fibrin sealant has a low production cost, does not transmit infectious diseases from human blood and has properties of a suitable scaffold for stem cells because it permits the preparation of differentiated scaffolds that are suitable for every need. PMID:24916098

Cytotoxicity assessments of Portulaca oleracea and Petroselinum sativum seed extracts on human hepatocellular carcinoma cells (HepG2).

PubMed

Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2014-01-01

The Pharmacological potential, such as antioxidant, anti-inflammatory, and antibacterial activities of Portulaca oleracea (PO) and Petroselinum sativum (PS) extracts are well known. However, the preventive properties against hepatocellular carcinoma cells have not been explored so far. Therefore, the present investigation was designed to study the anticancer activity of seed extracts of PO and PS on the human hepatocellular carcinoma cells (HepG2). The HepG2 cells were exposed with 5-500 μg/ml of PO and PS for 24 h. After the exposure, cell viability by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay, and cellular morphology by phase contrast inverted microscope were studied. The results showed that PO and PS extracts significantly reduced the cell viability of HepG2 in a concentration dependent manner. The cell viability was recorded to be 67%, 31%, 21%, and 17% at 50, 100, 250, and 500 μg/ml of PO, respectively by MTT assay and 91%, 62%, 27%, and 18% at 50, 100, 250, and 500 μg/ml of PO, respectively by NRU assay. PS exposed HepG2 cells with 100 μg/ml and higher concentrations were also found to be cytotoxic. The decrease in the cell viability at 100, 250, and 500 μg/ml of PS was recorded as 70%, 33%, and 15% by MTT assay and 63%, 29%, and 17%, respectively by NRU assay. Results also showed that PO and PS exposed cells reduced the normal morphology and adhesion capacity of HepG2 cells. HepG2 cells exposed with 50 μg/ml and higher concentrations of PO and PS lost their typical morphology, become smaller in size, and appeared in rounded bodies. Our results demonstrated preliminary screening of anticancer activity of Portulaca oleracea and Petroselinum sativum extracts against HepG2 cells, which can be further used for the development of a potential therapeutic anticancer agent.

Evaluation of radical scavenging activity, intestinal cell viability and antifungal activity of Brazilian propolis by-product.

PubMed

de Francisco, Lizziane; Pinto, Diana; Rosseto, Hélen; Toledo, Lucas; Santos, Rafaela; Tobaldini-Valério, Flávia; Svidzinski, Terezinha; Bruschi, Marcos; Sarmento, Bruno; Oliveira, M Beatriz P P; Rodrigues, Francisca

2018-03-01

Propolis is a natural adhesive resinous compound produced by honeybees to protect hives from bacteria and fungi, being extremely expensive for food industry. During propolis production, a resinous by-product is formed. This resinous waste is currently undervalued and underexploited. Accordingly, in this study the proximate physical and chemical quality, as well as the antioxidant activity, radical scavenging activity and cell viability of this by-product were evaluated and compared with propolis in order to boost new applications in food and pharmaceutical industries. The results revealed that the by-product meets the physical and chemical quality standards expected and showed that the propolis waste contains similar amounts of total phenolic content (TPC) and total flavonoid content (TFC) to propolis. Also, a good scavenging activity against reactive oxygen and nitrogen species (ROS and RNS, respectively) determined by the assays of superoxide anion radical (O 2 - ), hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HOCl), nitric oxide (NO) and peroxyl radical (ROO) were determined. Linear positive correlations were established between the TPC of both samples and the antioxidant activity evaluated by three different methods (DPPH, ABTS and FRAP assays). The extracts were also screened for cell viability assays in two different intestinal cell lines (HT29-MTX and Caco-2), showing a viability concentration-dependent. Similarly, the Artemia salina assay, used to assess toxicity, demonstrated the concentration influence on results. Finally, the antifungal activity against ATCC species of Candida was demonstrated. These results suggest that propolis by-product can be used as a new rich source of bioactive compounds for different areas, such as food or pharmaceutical. Copyright © 2017 Elsevier Ltd. All rights reserved.

MUC1-Targeted Cancer Cell Photothermal Ablation Using Bioinspired Gold Nanorods.

PubMed

Zelasko-Leon, Daria C; Fuentes, Christina M; Messersmith, Phillip B

2015-01-01

Recent studies have highlighted the overexpression of mucin 1 (MUC1) in various epithelial carcinomas and its role in tumorigenesis. These mucins present a novel targeting opportunity for nanoparticle-mediated photothermal cancer treatments due to their unique antenna-like extracellular extension. In this study, MUC1 antibodies and albumin were immobilized onto the surface of gold nanorods using a "primer" of polydopamine (PD), a molecular mimic of catechol- and amine-rich mussel adhesive proteins. PD forms an adhesive platform for the deposition of albumin and MUC1 antibodies, achieving a surface that is stable, bioinert and biofunctional. Two-photon luminescence confocal and darkfield scattering imaging revealed targeting of MUC1-BSA-PD-NRs to MUC1+ MCF-7 breast cancer and SCC-15 squamous cell carcinoma cells lines. Treated cells were exposed to a laser encompassing the near-infrared AuNR longitudinal surface plasmon and assessed for photothermal ablation. MUC1-BSA-PD-NRs substantially decreased cell viability in photoirradiated MCF-7 cell lines vs. MUC1- MDA-MB-231 breast cancer cells (p < 0.005). Agents exhibited no cytotoxicity in the absence of photothermal treatment. The facile nature of the coating method, combined with targeting and photoablation efficacy, are attractive features of these candidate cancer nanotherapeutics.

Effect of ellagic acid on proliferation, cell adhesion and apoptosis in SH-SY5Y human neuroblastoma cells.

PubMed

Fjaeraa, Christina; Nånberg, Eewa

2009-05-01

Ellagic acid, a polyphenolic compound found in berries, fruits and nuts, has been shown to possess growth-inhibiting and apoptosis promoting activities in cancer cell lines in vitro. The objective of this study was to investigate the effect of ellagic acid in human neuroblastoma SH-SY5Y cells. In cultures of SH-SY5Y cells incubated with ellagic acid, time- and concentration-dependent inhibitory effects on cell number were demonstrated. Ellagic acid induced cell detachment, decreased cell viability and induced apoptosis as measured by DNA strand breaks. Ellagic acid-induced alterations in cell cycle were also observed. Simultaneous treatment with all-trans retinoic acid did not rescue the cells from ellagic acid effects. Furthermore, the results suggested that pre-treatment with all-trans retinoic acid to induce differentiation and cell cycle arrest did not rescue the cells from ellagic acid-induced cell death.

Riboflavin deprivation inhibits macrophage viability and activity - a study on the RAW 264.7 cell line.

PubMed

Mazur-Bialy, Agnieszka Irena; Buchala, Beata; Plytycz, Barbara

2013-08-28

Riboflavin, or vitamin B2, as a precursor of the coenzymes FAD and FMN, has an indirect influence on many metabolic processes and determines the proper functioning of several systems, including the immune system. In the human population, plasma riboflavin concentration varies from 3·1 nM (in a moderate deficiency, e.g. in pregnant women) to 10·4 nM (in healthy adults) and 300 nM (in cases of riboflavin supplementation). The purpose of the present study was to investigate the effects of riboflavin concentration on the activity and viability of macrophages, i.e. on one of the immunocompetent cell populations. The study was performed on the murine monocyte/macrophage RAW 264.7 cell line cultured in medium with various riboflavin concentrations (3·1, 10·4, 300 and 531 nM). The results show that riboflavin deprivation has negative effects on both the activity and viability of macrophages and reduces their ability to generate an immune response. Signs of riboflavin deficiency developed in RAW 264.7 cells within 4 d of culture in the medium with a low riboflavin concentration (3·1 nM). In particular, the low riboflavin content reduced the proliferation rate and enhanced apoptotic cell death connected with the release of lactate dehydrogenase. The riboflavin deprivation impaired cell adhesion, completely inhibited the respiratory burst and slightly impaired phagocytosis of the zymosan particles. In conclusion, macrophages are sensitive to riboflavin deficiency; thus, a low riboflavin intake in the diet may affect the immune system and may consequently decrease proper host immune defence.

Vertically, interconnected carbon nanowalls as biocompatible scaffolds for osteoblast cells

NASA Astrophysics Data System (ADS)

Ion, Raluca; Vizireanu, Sorin; Luculescu, Catalin; Cimpean, Anisoara; Dinescu, Gheorghe

2016-07-01

The response of MC3T3-E1 pre-osteoblasts to vertically aligned, interconnected carbon nanowalls prepared by plasma enhanced chemical vapor deposition on silicon substrate has been evaluated in terms of cell adhesion, viability and cell proliferation. The behavior of osteoblasts seeded on carbon nanowalls was analyzed in parallel and compared with the behavior of the cells maintained in contact with tissue culture polystyrene (TCPS). The results demonstrate that osteoblasts adhere and remain viable in the long term on carbon nanowalls. Moreover, on the investigated scaffold cell proliferation was significantly promoted, although to a lower extent than on TCPS. Overall, the successful culture of osteoblasts on carbon nanowalls coated substrate confirms the biocompatibility of this scaffold, which could have potential applications in the development of orthopedic biomaterials.

Experimental self-etching HEMA-free adhesive systems: cytotoxicity and degree of conversion.

PubMed

Barbosa, Marília Oliveira; de Carvalho, Rodrigo Varella; Demarco, Flávio Fernando; Ogliari, Fabrício Aulo; Zanchi, Cesar Henrique; Piva, Evandro; da Silva, Adriana Fernandes

2015-01-01

The aim of this study was to evaluate the effect of replacing 2-hydroxyethyl methacrylate (HEMA) by methacrylate surfactant monomers on the cytotoxicity and degree of conversion of two-step self-etching dentin adhesive systems. Five HEMA-free adhesive systems were tested: Bis-EMA 10, Bis-EMA 30, PEG400, PEG400UDMA, PEG1000, and a HEMA group was used as positive control. The cytotoxicity of the experimental primers, with different monomer concentrations (2 or 20 wt%), and bond resins, containing 25 wt% surfactant, was assessed using murine fibroblast cell line 3T3 and the tetrazolium assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)). The degree of conversion of the bond resins was analyzed using Fourier transform infrared spectroscopy. The data were submitted to statistical analysis using level of significance set at P < 0.05. The PEG 1000 group obtained higher cell viability in comparison with HEMA in the 2 % primer. The cell survival rate using 20 % primer showed that PEG1000 and BIS-EMA 10 were less cytotoxic than HEMA. With regard to the eluate from bond resin, the data showed that the groups BIS-EMA 10, BIS-EMA 30 and PEG400UDMA were less cytotoxic than HEMA. No statistically significant difference was found among degrees of conversion of the experimental groups and HEMA. PEG 1000, BIS-EMA 10 and 30 monomers showed the biological potential for use in new adhesive system formulations since they showed lower cytotoxicity and similar degree of conversion when compared with the HEMA-containing group.

Mechanism and Anticancer Activity of the Metabolites of an Endophytic Fungi from Eucommia ulmoides Oliv.

PubMed

Li, Qi; Zhang, Yan; Shi, Jun-Ling; Wang, Yi-Lin; Zhao, Hao-Bin; Shao, Dong-Yan; Huang, Qing-Sheng; Yang, Hui; Jin, Ming-Liang

2017-01-01

Backgroud: Pinoresinol (Pin) and pinoresinol monoglucoside (PMG) are plant-derived lignan molecules with multiple functions. We showed previously that an endophytic fungus from Eucommia ulmoides Oliv., Phomopsis sp. XP-8 is able to produce Pin and PMG. This study was carried out to test the anti-tumor capability of the culture of XP-8 and identify the major effective compounds. The fungal culture was added in the culture of HepG2 and K562 cells, and the viabilities of these cells were detected and the possible mechanism was analyzed. The fungal culture showed significant capaiblity in decreasing the viability of tumor cells and induce apoptosis via up-regulation of the expression of apoptosis-related genes. It also significantly inhibited the adhesion and migration of HepG2 cells by blocking MMP-9 expression. Pin and PMG were isolated from the growth culture and shown to be the major effective components for inhibition. The study indicated the potential application of XP-8 in the production of anti-tumour products by the bioconversion of glucose. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Microfluidic Fabrication of Cell Adhesive Chitosan Microtubes

PubMed Central

Oh, Jonghyun; Kim, Keekyoung; Won, Sung Wook; Cha, Chaenyung; Gaharwar, Akhilesh; Selimović, Šeila; Bae, Hojae; Lee, Kwang Ho; Lee, Dong Hwan; Lee, Sang-Hoon; Khademhosseini, Ali

2013-01-01

Chitosan has been used as a scaffolding material in tissue engineering due to its mechanical properties and biocompatibility. With increased appreciation of the effect of micro- and nanoscale environments on cellular behavior, there is increased emphasis on generating microfabricated chitosan structures. Here we employed a microfluidic coaxial flow-focusing system to generate cell adhesive chitosan microtubes of controlled sizes by modifying the flow rates of a chitosan pre-polymer solution and phosphate buffered saline (PBS). The microtubes were extruded from a glass capillary with a 300 μm inner diameter. After ionic crosslinking with sodium tripolyphosphate (TPP), fabricated microtubes had inner and outer diameter ranges of 70-150 μm and 120-185 μm. Computational simulation validated the controlled size of microtubes and cell attachment. To enhance cell adhesiveness on the microtubes, we mixed gelatin with the chitosan pre-polymer solution and adjusted the pH values of the chitosan pre-polymer solution with gelatin and TPP. During the fabrication of microtubes, fibroblasts suspended in core PBS flow adhered to the inner surface of chitosan-gelatin microtubes. To achieve physiological pH values, we adjusted pH values of chiotsan pre-polymer solution and TPP. In particular, we were able to improve cell viability to 92% with pH values of 5.8 and 7.4 for chitosan and TPP solution respectively. Cell culturing for three days showed that the addition of the gelatin enhanced cell spreading and proliferation inside the chitosan-gelatin microtubes. The microfluidic fabrication method for ionically crosslinked chitosan microtubes at physiological pH can be compatible with a variety of cells and used as a versatile platform for microengineered tissue engineering. PMID:23355068

SLC12A7 alters adrenocortical carcinoma cell adhesion properties to promote an aggressive invasive behavior.

PubMed

Brown, Taylor C; Murtha, Timothy D; Rubinstein, Jill C; Korah, Reju; Carling, Tobias

2018-06-08

Altered expression of Solute Carrier Family 12 Member 7 (SLC12A7) is implicated to promote malignant behavior in multiple cancer types through an incompletely understood mechanism. Recent studies have shown recurrent gene amplifications and overexpression of SLC12A7 in adrenocortical carcinoma (ACC). The potential mechanistic effect(s) of SLC12A7 amplifications in portending an aggressive behavior in ACC has not been previously studied and is investigated here using two established ACC cell lines, SW-13 and NCI-H295R. SW-13 cells, which express negligible amounts of SLC12A7, were enforced to express SLC12A7 constitutively, while RNAi gene silencing was performed in NCI-H295R cells, which have robust endogenous expression of SLC12A7. In vitro studies tested the outcomes of experimental alterations in SLC12A7 expression on malignant characteristics, including cell viability, growth, colony formation potential, motility, invasive capacity, adhesion and detachment kinetics, and cell membrane organization. Further, potential alterations in transcription regulation downstream to induced SLC12A7 overexpression was explored using targeted transcription factor expression arrays. Enforced SLC12A7 overexpression in SW-13 cells robustly promoted motility and invasive characteristics (p < 0.05) without significantly altering cell viability, growth, or colony formation potential. SLC12A7 overexpression also significantly increased rates of cellular attachment and detachment turnover (p < 0.05), potentially propelled by increased filopodia formation and/or Ezrin interaction. In contrast, RNAi gene silencing of SLC12A7 stymied cell attachment strength as well as migration and invasion capacity in NCI-H295R cells. Transcription factor expression analysis identified multiple signally pathways potentially affected by SLC12A7 overexpression, including osmotic stress, bone morphogenetic protein, and Hippo signaling pathways. Amplification of SLC12A7 observed in ACCs is shown here, in vitro, to exacerbate the malignant behavior of ACC cells by promoting invasive capacities, possibly mediated by alterations in multiple signaling pathways, including the osmotic stress pathway.

JAM-A as a prognostic factor and new therapeutic target in multiple myeloma

PubMed Central

Solimando, A G; Brandl, A; Mattenheimer, K; Graf, C; Ritz, M; Ruckdeschel, A; Stühmer, T; Mokhtari, Z; Rudelius, M; Dotterweich, J; Bittrich, M; Desantis, V; Ebert, R; Trerotoli, P; Frassanito, M A; Rosenwald, A; Vacca, A; Einsele, H; Jakob, F; Beilhack, A

2018-01-01

Cell adhesion in the multiple myeloma (MM) microenvironment has been recognized as a major mechanism of MM cell survival and the development of drug resistance. Here we addressed the hypothesis that the protein junctional adhesion molecule-A (JAM-A) may represent a novel target and a clinical biomarker in MM. We evaluated JAM-A expression in MM cell lines and in 147 MM patient bone marrow aspirates and biopsies at different disease stages. Elevated JAM-A levels in patient-derived plasma cells were correlated with poor prognosis. Moreover, circulating soluble JAM-A (sJAM-A) levels were significantly increased in MM patients as compared with controls. Notably, in vitro JAM-A inhibition impaired MM migration, colony formation, chemotaxis, proliferation and viability. In vivo treatment with an anti-JAM-A monoclonal antibody (αJAM-A moAb) impaired tumor progression in a murine xenograft MM model. These results demonstrate that therapeutic targeting of JAM-A has the potential to prevent MM progression, and lead us to propose JAM-A as a biomarker in MM, and sJAM-A as a serum-based marker for clinical stratification. PMID:29064484

The use of magnetron sputtering for the deposition of thin titanium coatings on the surface of bioresorbable electrospun fibrous scaffolds for vascular tissue engineering: A pilot study

NASA Astrophysics Data System (ADS)

Bolbasov, E. N.; Antonova, L. V.; Stankevich, K. S.; Ashrafov, A.; Matveeva, V. G.; Velikanova, E. A.; Khodyrevskaya, Yu. I.; Kudryavtseva, Yu. A.; Anissimov, Y. G.; Tverdokhlebov, S. I.; Barbarash, L. S.

2017-03-01

The deposition of thin titanium coatings using magnetron spattering on the surface of bioresorbable fibrous scaffolds produced by electrospinning was investigated. Parameters that allow the surface modification without damaging the "macro" structure of scaffolds were determined. Physicochemical properties of the modified scaffolds were described using SEM, EDS, DSC, optical goniometry, and mechanical testing. It was shown that plasma treatment has a significant influence on the scaffolds' fiber surface relief. The modification process leads to a slight decrease of the scaffold mechanical performance mainly caused by polymer crystallization. Increasing the deposition time increases the amount of titanium on the surface. The biocompatibility of the modified scaffolds was studied using hybridoma of the endothelial cells of human umbilical vein and human lung carcinoma (EA.hy 926 cell line). Cell adhesion, viability, and secretion of interleukin-6 (IL6), interleukin-8 (IL8), and vascular endothelial growth factor (VEGF) were investigated. It was demonstrated that the deposition of thin titanium coatings on the fibrous scaffolds' surface enhances cell adhesion. Additionally, it was determined that modified scaffolds have proangiogenic activity.

Matrix metalloproteases inhibition and biocompatibility of gold and platinum nanoparticles.

PubMed

Hashimoto, Masanori; Kawai, Koji; Kawakami, Hayato; Imazato, Satoshi

2016-01-01

Matrix metalloprotease (MMP) inhibitors improve the longevity of dental adhesives/tooth bonds; however, biocompatibility is required for their clinical use. This study evaluated the inhibition of MMPs and toxicity of two gold (AuNPs) and platinum nanoparticles (PtNPs) as possible compounds for use in dental adhesives. The MMP assay for studying the interaction of MMPs and nanoparticles (NPs) was evaluated by an MMP assay kit and gelatin zymography. Cultured L929 fibroblast cells or RAW264 macrophages were exposed to NPs. The cellular responses to NPs were examined using cytotoxic (cell viability) and genotoxic assays (comet assay), and transmission electron microscopic (TEM) analysis. The mechanical properties (elastic modulus) of the experimental resin loaded with NPs were examined using thermomechanical analysis. All NPs inhibited MMP activity at relatively low concentrations. The NPs inhibit MMPs by chelating with the Zn(2+) bound in the active sites of MMPs. No cytotoxic and genotoxic effects were found in AuNPs, whereas the PtNPs possessed both adverse effects. In TEM analysis, the NPs were localized mainly in lysosomes without penetration into nuclei. The mechanical properties of the resins increased when AuNPs were added in resins, but not by PtNPs. AuNPs are attractive candidates to inhibit MMPs and improve the mechanical properties of resins without cytotoxic/genotoxic effects to cells, and therefore should be suitable for applications in adhesive resin systems. © 2015 Wiley Periodicals, Inc.

Chemically grafted fibronectin for use in QCM-D cell studies

PubMed Central

Sobolewski, Peter; Tomczyk, Nancy; Composto, Russell J.; Eckmann, David M.

2014-01-01

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using surface ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics. PMID:24657645

Three-dimensional printed bone scaffolds: The role of nano/micro-hydroxyapatite particles on the adhesion and differentiation of human mesenchymal stem cells.

PubMed

Domingos, Marco; Gloria, Antonio; Coelho, Jorge; Bartolo, Paulo; Ciurana, Joaquim

2017-06-01

Bone tissue engineering is strongly dependent on the use of three-dimensional scaffolds that can act as templates to accommodate cells and support tissue ingrowth. Despite its wide application in tissue engineering research, polycaprolactone presents a very limited ability to induce adhesion, proliferation and osteogenic cell differentiation. To overcome some of these limitations, different calcium phosphates, such as hydroxyapatite and tricalcium phosphate, have been employed with relative success. This work investigates the influence of nano-hydroxyapatite and micro-hydroxyapatite (nHA and mHA, respectively) particles on the in vitro biomechanical performance of polycaprolactone/hydroxyapatite scaffolds. Morphological analysis performed with scanning electron microscopy allowed us to confirm the production of polycaprolactone/hydroxyapatite constructs with square interconnected pores of approximately 350 µm and to assess the distribution of hydroxyapatite particles within the polymer matrix. Compression mechanical tests showed an increase in polycaprolactone compressive modulus ( E) from 105.5 ± 11.2 to 138.8 ± 12.9 MPa (PCL_nHA) and 217.2 ± 21.8 MPa (PCL_mHA). In comparison to PCL_mHA scaffolds, the addition of nano-hydroxyapatite enhanced the adhesion and viability of human mesenchymal stem cells as confirmed by Alamar Blue assay. In addition, after 14 days of incubation, PCL_nHA scaffolds showed higher levels of alkaline phosphatase activity compared to polycaprolactone or PCL_mHA structures.

A comparison inhibitory effects of cisplatin and MNPs-PEG-cisplatin on the adhesion capacity of bone metastatic breast cancer.

PubMed

Mokhtari, Mohammad Javad; Koohpeima, Fatemeh; Mohammadi, Hadi

2017-10-01

To date, high mortality in women due to malignancy breast cancer related to the metastasis to the bone is a significant challenge. As, magnetic nanoparticles (MNPs) conjugated with the biocompatible polymers was employed for the delivery of some hydrophobic anticancer agents, the main aim of the current research was to assess whether cisplatin-loaded MNPs enhanced the anticancer effect of free cisplatin in breast cancer cells. MNPs decorated with PEG were synthesized by an improved coprecipitation technique, and then cisplatin was loaded onto the MNPs via a simple mixing method. Afterward, its morphology, size, chemical structure, magnetic property, hydrodynamic diameter, zeta potential, and crystal structure were characterized by scanning and transmittance electron microscopy, Fourier transforms infrared spectroscopy, vibrating sample magnetometer, dynamic light scattering, and X-ray powder diffraction and flame atomic absorption spectroscopy respectively. Additionally, the effects of cisplatin and MNPs-PEG-cisplatin on viability, migration and adhesion capacity of T47D cells were investigated by evaluating α2-integrin and β1-integrin; mRNAs were assessed by real-time RT-PCR. Consequently, the in vitro assay results showed a considerable dose-dependent inhibitory effect of cisplatin and MNPs-PEG-cisplatin on proliferation, migration, and adhesion of T47D cells. Finally, current research was shown that MNPs-PEG-cisplatin strongly increased anticancer effects compared with free cisplatin in the T47D cell line. © 2017 John Wiley & Sons A/S.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

PubMed

Kikuchi, Hidetomo; Yuan, Bo; Nishimura, Yoshio; Imai, Masahiko; Furutani, Ryota; Kamoi, Saki; Seno, Misako; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Hu, Xiao-Mei; Takagi, Norio; Hirano, Toshihiko; Toyoda, Hiroo

2013-12-01

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D₃ (VD₃)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Transportation Conditions for Prompt Use of Ex Vivo Expanded and Freshly Harvested Clinical-Grade Bone Marrow Mesenchymal Stromal/Stem Cells for Bone Regeneration

PubMed Central

Veronesi, Elena; Murgia, Alba; Caselli, Anna; Grisendi, Giulia; Piccinno, Maria Serena; Rasini, Valeria; Giordano, Rosaria; Montemurro, Tiziana; Bourin, Philippe; Sensebé, Luc; Rojewski, Markus T.; Schrezenmeier, Hubert; Layrolle, Pierre; Ginebra, Maria Pau; Panaitescu, Carmen Bunu; Gómez-Barrena, Enrique; Catani, Fabio; Paolucci, Paolo; Burns, Jorge S.

2014-01-01

Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9% saline alone or with 4% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/β-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/β-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/β-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4% HSA in 0.9% saline to a destination 18 h away showed prompt adhesion to HA/β-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine. PMID:23845029

Hydroxyapatite nanobelt/polylactic acid Janus membrane with osteoinduction/barrier dual functions for precise bone defect repair.

PubMed

Ma, Baojin; Han, Jing; Zhang, Shan; Liu, Feng; Wang, Shicai; Duan, Jiazhi; Sang, Yuanhua; Jiang, Huaidong; Li, Dong; Ge, Shaohua; Yu, Jinghua; Liu, Hong

2018-04-15

Controllable osteoinduction maintained in the original defect area is the key to precise bone repair. To meet the requirement of precise bone regeneration, a hydroxyapatite (HAp) nanobelt/polylactic acid (PLA) (HAp/PLA) Janus membrane has been successfully prepared in this study by coating PLA on a paper-like HAp nanobelt film by a casting-pervaporation method. The Janus membrane possesses dual functions: excellent osteoinduction from the hydrophilic HAp nanobelt side and barrier function originating from the hydrophobic PLA film. The cell viability and osteogenic differentiation ability of human adipose-derived stem cells (hADSCs) on the Janus membrane were assessed. The in vitro experimental results prove that the HAp nanobelt side presents high cell viability and efficient osteoinduction without any growth factor and that the PLA side can prohibit cell attachment. The in vivo repair experiments on a rat mandible defect model prove that the PLA side can prevent postoperative adhesion between bone and adjacent soft tissues. Most importantly, the HAp side has a strong ability to promote defect repair and bone regeneration. Therefore, the HAp/PLA Janus membrane will have wide applications as a kind of tissue engineering material in precise bone repair because of its unique dual osteoinduction/barrier functions, biocompatibility, low cost, and its ability to be mass-produced. Precise bone defect repair to keeping tissue integrity and original outline shape is a very important issue for tissue engineering. Here, we have designed and prepared a novel HAp/PLA Janus membrane using a casting-pervaporation method to form a layer of PLA film on paper-like HAp nanobelt film. HAp nanobelt side of the Janus membrane can successfully promote osteogenic differentiation. PLA side of the Janus membrane exhibits good properties as a barrier for preventing the adhesion of cells in vitro. Mandible repair experiments in vivo have shown that the HAp/PLA Janus membrane can promote rat mandible repair on the HAp side and can successfully prevent postoperative adhesion on the PLA side at the same time. Therefore, the HAp/PLA Janus membrane with its osteoinduction/barrier dual functions can be applied to repair bone defect precisely. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biological Behavior of Osteoblast Cell and Apatite Forming Ability of the Surface Modified Ti Alloys.

PubMed

Zhao, Jingming; Hwang, K H; Choi, W S; Shin, S J; Lee, J K

2016-02-01

Titanium as one kind of biomaterials comes in direct contact with the body, making evaluation of biocompatibility an important aspect to biomaterials development. Surface chemistry of titanium plays an important role in osseointegration. Different surface modification alters the surface chemistry and result in different biological response. In this study, three kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coating successfully formed on the Ti surfaces in simulated body fluid. Strong mixed acid increased the roughness of the metal surface, because the porous and rough surface allows better adhesion between Ca-P coatings and substrates. After modification of titanium surface by mixed acidic solution and subsequently H2O2/HCL treatment evaluation of biocompatibility was conducted from hydroxyapatite formation by biomimetic process and cell viability on modified titanium surface. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Results from this study indicated that surface treatment methods affect the surface morphology, type of TiO2 layer formed and subsequent apatite deposition and biological responses. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometric/colorimetric growth indicator, without any toxic and side effect to cell line. In addition, mixed acid treatment uses a lower temperature and shorter time period than widely used alkali treatment.

Synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK inhibitors with anti-angiogenic activity.

PubMed

Dao, Pascal; Jarray, Rafika; Le Coq, Johanne; Lietha, Daniel; Loukaci, Ali; Lepelletier, Yves; Hadj-Slimane, Réda; Garbay, Christiane; Raynaud, Françoise; Chen, Huixiong

2013-08-15

We report herein the synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK (focal adhesion kinase) inhibitors and the evaluation of their anti-angiogenic activity on HUVEC cells. Generally, the effects of these compounds on endothelial cells could be correlated with their kinase inhibitory activity. The most efficient compounds displayed inhibition of viability against HUVEC cells in the micromolar range, as observed with TAE-226, which was designed by Novartis Pharma AG. X-ray crystallographic analysis of the co-crystal structure for compound 34 revealed that the mode of interaction with the FAK kinase domain is highly similar to that observed in the complex of TAE-226. Copyright © 2013 Elsevier Ltd. All rights reserved.

Photochemically modified diamond-like carbon surfaces for neural interfaces.

PubMed

Hopper, A P; Dugan, J M; Gill, A A; Regan, E M; Haycock, J W; Kelly, S; May, P W; Claeyssens, F

2016-01-01

Diamond-like carbon (DLC) was modified using a UV functionalization method to introduce surface-bound amine and aldehyde groups. The functionalization process rendered the DLC more hydrophilic and significantly increased the viability of neurons seeded to the surface. The amine functionalized DLC promoted adhesion of neurons and fostered neurite outgrowth to a degree indistinguishable from positive control substrates (glass coated with poly-L-lysine). The aldehyde-functionalized surfaces performed comparably to the amine functionalized surfaces and both additionally supported the adhesion and growth of primary rat Schwann cells. DLC has many properties that are desirable in biomaterials. With the UV functionalization method demonstrated here it may be possible to harness these properties for the development of implantable devices to interface with the nervous system. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Neural Differentiation of Mesenchymal Stem Cells on Scaffolds for Nerve Tissue Engineering Applications.

PubMed

Quintiliano, Kerlin; Crestani, Thayane; Silveira, Davi; Helfer, Virginia Etges; Rosa, Annelise; Balbueno, Eduardo; Steffens, Daniela; Jotz, Geraldo Pereira; Pilger, Diogo André; Pranke, Patricia

2016-11-01

Scaffolds produced by electrospinning act as supports for cell proliferation and differentiation, improved through the release of neurotrophic factors. The objective of this study was to develop aligned and random nanofiber scaffolds with and without nerve growth factor to evaluate the potential of mesenchymal stem cells (MSCs) for neural differentiation. Nanofiber morphology, diameter, degradability, cell morphology, adhesion, proliferation, viability, cytotoxicity, and neural differentiation were performed to characterize the scaffolds. The expression for nestin, β-III tubulin, and neuron-specific enolase was also evaluated. The scaffolds demonstrated a satisfactory environment for MSC growth, being nontoxic. The MSCs cultivated on the scaffolds were able to adhere and proliferate. The evaluation of neural differentiation indicated that in all groups of scaffolds the MSCs were able to upregulate neural gene expression.

Production and in vitro evaluation of soy protein-based biofilms as a support for human keratinocyte and fibroblast culture.

PubMed

Curt, Sèverine; Subirade, Muriel; Rouabhia, Mahmoud

2009-06-01

This study presents results on soy protein isolate (SPI) biofilm production and the corresponding effect on the stability and toxicity of the derived films. SPI biofilms were prepared from SPI chemically treated with formaldehyde at various concentrations (0%, 1%, 2%, and 3%) as cross-linking agents. In vitro SPI biofilm degradation was evaluated as a function of water absorption leading to weight and size modifications. SPI biofilm toxicity was determined as a function of human keratinocyte and fibroblast adhesion, viability, and proliferation. Cytokine gene expression supported this using reverse transcriptase polymerase chain reaction techniques. Our results confirm that SPI can be used to produce biofilms. The resulting SPI biofilms without formaldehyde swell significantly, which leads to their physical instability. Formaldehyde treatment enhanced the mechanical properties of these biofilms by covalently cross-linking polypeptide chains. The decreased water absorption was dependent on the amount of formaldehyde present. SPI biofilms with 2% and 3% formaldehyde were highly stable and easier to manipulate than those with 0% and 1% formaldehyde. Tissue culture analyses revealed that the SPI biofilms without formaldehyde were non-toxic to human cells (keratinocytes and fibroblasts). The presence of formaldehyde in biofilms did not have any effects on cell viability, adhesion, or proliferation. This was supported by the high level of messenger RNA expression of interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha by the keratinocytes and of IL-6 and IL-8 by the fibroblasts. Overall, we produced a stable, non-toxic soy protein support, which may be of potential interest in medical applications such as cell culture matrices and damaged tissue replacement.

Improvement of biomaterials used in tissue engineering by an ageing treatment.

PubMed

Acevedo, Cristian A; Díaz-Calderón, Paulo; Enrione, Javier; Caneo, María J; Palacios, Camila F; Weinstein-Oppenheimer, Caroline; Brown, Donald I

2015-04-01

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60% (scaffold without ageing treatment) to 80%. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.

Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

PubMed Central

Shahid, Muhammad; Wang, Jianfang; Gu, Xiaolong; Chen, Wei; Ali, Tariq; Gao, Jian; Han, Dandan; Yang, Rui; Fanning, Séamus; Han, Bo

2017-01-01

Prototheca zopfii infections are becoming global concerns in humans and animals. Bovine protothecal mastitis is characterized by deteriorating milk quality and quantity, thus imparting huge economic losses to dairy industry. Previous published studies mostly focused on the prevalence and characterization of P. zopfii from mastitis. However, the ultrastructural pathomorphological changes associated with apoptosis in bovine mammary epithelial cells (bMECs) are not studied yet. Therefore, in this study we aimed to evaluate the in vitro comparative apoptotic potential of P. zopfii genotype-I and -II on bMECs using flow cytometry, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results showed fast growth rate and higher adhesion capability of genotype-II in bMECs as compared with genotype-I. The viability of bMECs infected with P. zopfii genotype-II was significantly decreased after 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control cells. Contrary, genotype-I couldn't show any significant effects on cell viability. Moreover, after infection of bMECs with genotype-II, the apoptosis increased significantly at 12 h (p < 0.05) and 24 h (p < 0.01) as compared with control group. Genotype-I couldn't display any significant effects on cell apoptosis. The host specificity of P. zopfii was also tested in mouse osteoblast cells, and the results suggest that genotype-I and -II could not cause any significant apoptosis in these cell lines. SEM interpreted the pathomorphological alterations in bMECs after infection. Adhesion of P. zopfii with cells and further disruption of cytomembrane validated the apoptosis caused by genotype-II under SEM. While genotype-1 couldn't cause any significant apoptosis in bMECs. Furthermore, genotype-II induced apoptotic manifested specific ultrastructure features, like cytoplasmic cavitation, swollen mitochondria, pyknosis, cytomembrane disruption, and appearance of apoptotic bodies under TEM. The findings of the current study revealed that genotype-II has the capability to invade and survive within the bMECs, thus imparting significant damages to the mammary cells which result in apoptosis. This study represents the first insights into the pathomorphological and ultrastructure features of apoptosis in bMECs induced by P. zopfii genotype-II. PMID:28752077

Fundus autofluorescence and fate of glycoxidized particles injected into subretinal space in rabbit age-related macular degeneration model.

PubMed

Hirata, Megumi; Yasukawa, Tsutomu; Wiedemann, Peter; Kimura, Erika; Kunou, Noriyuki; Eichler, Wolfram; Takase, Ayae; Sato, Rina; Ogura, Yuichiro

2009-07-01

Abnormal fundus autofluorescence (FAF) is associated with the incidence or progression of dry and wet age-related macular degeneration (AMD). We previously developed a rabbit AMD model with drusen and type-1 choroidal neovascularization (CNV) that mimics the accumulation of lipofuscin using artificial glycoxidized particles. The objective of the current study was to investigate in vitro effects of glycoxidized particles on retinal pigment epithelial (RPE) cells, and the FAF and fate of injected particles in this model. Glycoxidized particles were prepared by a 4-day incubation of water-in-oil emulsions of serum albumin and glycolaldehyde to allow glycoxidation and consequent cross-linking. After particles were added in the culture medium of confluent human RPE cells, cell viability, adhesion activity, and proliferation activity were assessed by cell counting. In anesthetized rabbits, 250 microg of glycoxidized particles were injected into the subretinal space to induce experimental AMD. FAF measurement and angiography with sodium fluorescein and indocyanine green were performed periodically using the Heidelberg Retina Angiograph 2 (HRA2). The eyes enucleated, and the lung and the spleen, excised at week 4 or 12, were histologically evaluated by light and fluorescence microscopy. Glycoxidized particles phagocytosed did not impair the cell viability, adhesion, and proliferation of RPE cells, as compared with RPE cells in controls. HRA2 showed different patterns of abnormal FAF in the area with the implanted glycoxidized particles, similar to pathological FAF patterns in aging human eyes with or without AMD. Histologic examination showed accumulated glycoxidized particles and large lipofuscin granules with green autofluorescence in and under the RPE and at the margins of or beneath drusen, possibly associated with abnormal FAF. In addition, some particles were detected in the lung and the spleen. Glycoxidized particles phagocytosed might stay in RPE cells without any acute biological reaction. Our rabbit model of AMD simulated abnormal FAF patterns observed in aging human eyes with or without AMD. Glycoxidized particles phagocytosed by RPE cells could be deposited on Bruch's membrane in rabbits, possibly excreted in part into choroidal circulation. This model may be useful for understanding various patterns of abnormal FAF histologically, and for elucidating the pathogenesis of AMD.

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

PubMed Central

1989-01-01

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern- regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM. PMID:2473081

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats.

PubMed

Enami, Yuta; Joseph, Brigid; Bandi, Sriram; Lin, Juan; Gupta, Sanjeev

2012-04-01

Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications. Copyright © 2012 American Association for the Study of Liver Diseases.

The Compatibility of Hepatocytes with Chemically Modified Porous Silicon with Reference to In Vitro Biosensors

PubMed Central

Alvarez, Sara D.; Derfus, Austin M.; Schwartz, Michael P.; Bhatia, Sangeeta N.; Sailor, Michael J.

2008-01-01

Porous Si is a nanostructured material that is of interest for molecular and cell-based biosensing, drug delivery, and tissue engineering applications. Surface chemistry is an important factor determining the stability of porous Si in aqueous media, its affinity for various biomolecular species, and its compatibility with tissues. In this study, the attachment and viability of a primary cell type to porous Si samples containing various surface chemistries is reported, and the ability of the porous Si films to retain their optical reflectivity properties relevant to molecular biosensing is assessed. Four chemical species grafted to the porous Si surface are studied: silicon oxide (via ozone oxidation), dodecyl (via hydrosilylation with dodecene), undecanoic acid (via hydrosilylation with undecylenic acid), and oligo(ethylene) glycol (via hydrosilylation with undecylenic acid followed by an oligo(ethylene) glycol coupling reaction). Fourier Transform Infrared (FTIR) spectroscopy and contact angle measurements are used to characterize the surface. Adhesion and short-term viability of primary rat hepatocytes on these surfaces, with and without pre-adsorption of collagen type I, are assessed using vital dyes (calcein-AM and ethidium homodimer I). Cell viability on undecanoic acid-terminated porous Si, oxide-terminated porous Si, and oxide-terminated flat (non-porous) Si are monitored by quantification of albumin production over the course of 8 days. The stability of porous Si thin films after 8 days in cell culture is probed by measuring the optical interferometric reflectance spectra. Results show that hepatocytes adhere better to surfaces coated with collagen, and that chemical modification does not exert a deleterious effect on primary rat hepatocytes. The hydrosilylation chemistry greatly improves the stability of porous Si in contact with cultured primary cells while allowing cell coverage levels comparable to standard culture preparations on tissue culture polystyrene. PMID:18845334

Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays

PubMed Central

Johnen, Sandra; Meißner, Frank; Krug, Mario; Baltz, Thomas; Endler, Ingolf; Mokwa, Wilfried; Walter, Peter

2016-01-01

Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue. PMID:27200182

Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays.

PubMed

Johnen, Sandra; Meißner, Frank; Krug, Mario; Baltz, Thomas; Endler, Ingolf; Mokwa, Wilfried; Walter, Peter

2016-01-01

Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue.

Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate

PubMed Central

Pourmollaabbassi, Babak; Karbasi, Saeed; Hashemibeni, Batool

2016-01-01

Background: The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Materials and Methods: In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. Results: The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio2) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. Conclusions: It is likely that the relation between HA and TiO2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface. PMID:27761431

Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate.

PubMed

Pourmollaabbassi, Babak; Karbasi, Saeed; Hashemibeni, Batool

2016-01-01

The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio 2 ) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO 2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. It is likely that the relation between HA and TiO 2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface.

Expression of immunoglobulin G in human podocytes, and its role in cell viability and adhesion.

PubMed

Jing, Ziyang; Deng, Hui; Ma, Junfan; Guo, Yanhong; Liang, Yaoxian; Wu, Rui; A, Lata; Geng, Zihan; Qiu, Xiaoyan; Wang, Yue

2018-06-01

Podocyte injury occurs during the initiation and development of numerous forms of glomerular disease, and antibodies targeting podocytes have become a biomarker for diagnosis and monitoring treatment response. Accumulating evidence has suggested that immunoglobulin (Ig) is expressed in non‑B lineage cells, including epithelial cancer cells, myeloid cells and several types of normal cells. The main aim of the present study was to ascertain the expression of IgG in human podocytes and to determine its potential role in cellular bioactivity. The present study detected positive staining for IgG heavy chain (Igγ) and its subtype γ4, and the light chains κ and λ in the cytoplasm or on the membrane by immunofluorescence. In addition, positive bands were detected for Igγ, γ1, γ3, γ4, κ and λ in the lysates of a podocyte cell line by western blotting. Mass spectrometry confirmed IgG1 as an intact tetramer in the culture supernatant. Constant region transcripts of Igγ, γ1, γ3, γ4, κ and λ were identified by reverse transcription‑polymerase chain reaction, and DNA sequencing of these transcripts revealed 96‑99% similarity with Ig mRNAs in the National Center for Biotechnology Information database. Compared with the diverse gene rearrangements from B cell-derived Ig, podocyte‑derived Ig exhibited conservative V(D)J patterns in the variable regions of Igγ and κ chains. Furthermore, the present study investigated the mechanism underlying IgG production in these cells by examining the expression of recombination activating gene (RAG)1, RAG2 and activation‑induced cytidine deaminase. The expression levels of these proteins suggested that podocyte‑derived Ig and traditional Ig may be generated in a similar manner. Furthermore, small interfering RNA‑mediated downregulation of IgG expression reduced podocyte viability and adhesive capabilities. These findings suggested that IgG is expressed in podocytes and that this expression may be associated with podocyte function. Due to its potential biological and clinical significance, this phenomenon warrants further investigation.

Layer-by-layer buildup of polysaccharide-containing films: Physico-chemical properties and mesenchymal stem cells adhesion.

PubMed

Kulikouskaya, Viktoryia I; Pinchuk, Sergei V; Hileuskaya, Kseniya S; Kraskouski, Aliaksandr N; Vasilevich, Irina B; Matievski, Kirill A; Agabekov, Vladimir E; Volotovski, Igor D

2018-03-22

Layer-by-Layer assembled polyelectrolyte films offer the opportunity to control cell attachment and behavior on solid surfaces. In the present study, multilayer films based on negatively charged biopolymers (pectin, dextran sulfate, carboxymethylcellulose) and positively charged polysaccharide chitosan or synthetic polyelectrolyte polyethyleneimine has been prepared and evaluated. Physico-chemical properties of the formed multilayer films, including their growth, morphology, wettability, stability, and mechanical properties, have been studied. We demonstrated that chitosan-containing films are characterized by the linear growth, the defect-free surface, and predominantly viscoelastic properties. When chitosan is substituted for the polyethyleneimine in the multilayer system, the properties of the formed films are significantly altered: the rigidity and surface roughness increases, the film growth acquires the exponential character. The multilayer films were subsequently used for culturing mesenchymal stem cells. It has been determined that stem cells effectively adhered to chitosan-containing films and formed on them the monolayer culture of fibroblast-like cells with high viability. Our results show that cell attachment is a complex process which is not only governed by the surface functionality because one of the key parameter effects on cell adhesion is the stiffness of polyelectrolyte multilayer films. We therefore propose our Layer-by-Layer films for applications in tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2018. © 2018 Wiley Periodicals, Inc.

Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway.

PubMed

Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

2016-11-07

Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal-regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway.

Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

PubMed Central

Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

2016-01-01

Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

PubMed

Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

2016-09-01

Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016. © 2016 Wiley Periodicals, Inc.

Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues

PubMed Central

Han, Bumsoo; Teo, Ka Yaw; Ghosh, Soham; Dutton, J. Craig; Grinnell, Frederick

2012-01-01

Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters – i) freezing temperature and corresponding cooling rate; and ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs. PMID:23246556

Biofunctionalized Lysophosphatidic Acid/Silk Fibroin Film for Cornea Endothelial Cell Regeneration

PubMed Central

Jeon, Hayan; Oliveira, Joaquim Miguel; Reis, Rui Luis; Khang, Gilson

2018-01-01

Cornea endothelial cells (CEnCs) tissue engineering is a great challenge to repair diseased or damaged CEnCs and require an appropriate biomaterial to support cell proliferation and differentiation. Biomaterials for CEnCs tissue engineering require biocompatibility, tunable biodegradability, transparency, and suitable mechanical properties. Silk fibroin-based film (SF) is known to meet these factors, but construction of functionalized graft for bioengineering of cornea is still a challenge. Herein, lysophosphatidic acid (LPA) is used to maintain and increase the specific function of CEnCs. The LPA and SF composite film (LPA/SF) was fabricated in this study. Mechanical properties and in vitro studies were performed using a rabbit model to demonstrate the characters of LPA/SF. ATR-FTIR was characterized to identify chemical composition of the films. The morphological and physical properties were performed by SEM, AFM, transparency, and contact angle. Initial cell density and MTT were performed for adhesion and cell viability in the SF and LPA/SF film. Reverse transcription polymerase chain reactions (RT-PCR) and immunofluorescence were performed to examine gene and protein expression. The results showed that films were designed appropriately for CEnCs delivery. Compared to pristine SF, LPA/SF showed higher biocompatibility, cell viability, and expression of CEnCs specific genes and proteins. These indicate that LPA/SF, a new biomaterial, offers potential benefits for CEnCs tissue engineering for regeneration. PMID:29710848

Endothelial responses of magnesium and other alloying elements in magnesium-based stent materials

PubMed Central

Zhao, Nan; Zhu, Donghui

2016-01-01

Biodegradable tailored magnesium (Mg) alloys are some of the most promising scaffolds for cardiovascular stents. During the course of degradation after implantation, all the alloying elements in the scaffold will be released to the surrounding vascular tissues. However, fundamental questions regarding the toxicity of alloying elements towards vascular cells, the maximum amount of each element that could be used in alloy design, or how each of the alloying elements affects vascular cellular activity and gene expression, are still not fully answered. This work systematically addressed these questions by revealing how application of different alloying elements commonly used in Mg stent materials influences several indices of human endothelial cell health, i.e., viability, proliferations, cytoskeletal reorganizations, migration, and the gene expression profile. The overall cell viability and proliferation showed a decreasing trend with increasing concentrations of the ions, and the half maximal effective concentrations (EC50) for each element were determined. When applied at a low concentration of around 10 mM, Mg had no adverse effects but improved cell proliferation and migration instead. Mg ions also altered endothelial gene expression significantly in a dose dependent manner. Most of the changed genes are related to angiogenesis and the cell adhesion signaling pathways. Findings from this work provide useful information on maximum safe doses of these ions for endothelial cells, endothelial responses towards these metal ions, and some guidance for future Mg stent design. PMID:25363018

Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth

NASA Astrophysics Data System (ADS)

Singh, Nandita; Chen, Jinhu; Koziol, Krzysztof K.; Hallam, Keith R.; Janas, Dawid; Patil, Avinash J.; Strachan, Ally; G. Hanley, Jonathan; Rahatekar, Sameer S.

2016-04-01

The design of biocompatible implants for neuron repair/regeneration ideally requires high cell adhesion as well as good electrical conductivity. Here, we have shown that plasma-treated chitin carbon nanotube composite scaffolds show very good neuron adhesion as well as support of synaptic function of neurons. The addition of carbon nanotubes to a chitin biopolymer improved the electrical conductivity and the assisted oxygen plasma treatment introduced more oxygen species onto the chitin nanotube scaffold surface. Neuron viability experiments showed excellent neuron attachment onto plasma-treated chitin nanotube composite scaffolds. The support of synaptic function was evident on chitin/nanotube composites, as confirmed by PSD-95 staining. The biocompatible and electrically-conducting chitin nanotube composite scaffold prepared in this study can be used for in vitro tissue engineering of neurons and, potentially, as an implantable electrode for stimulation and repair of neurons.

Preparation and characterization of a novel skin substitute.

PubMed

Castagnoli, Carlotta; Fumagalli, Mara; Alotto, Daniela; Cambieri, Irene; Casarin, Stefania; Ostorero, Alessia; Casimiri, Raffaella; Germano, Patrizia; Pezzuto, Carla; Stella, Maurizio

2010-01-01

Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.

Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth.

PubMed

Singh, Nandita; Chen, Jinhu; Koziol, Krzysztof K; Hallam, Keith R; Janas, Dawid; Patil, Avinash J; Strachan, Ally; G Hanley, Jonathan; Rahatekar, Sameer S

2016-04-21

The design of biocompatible implants for neuron repair/regeneration ideally requires high cell adhesion as well as good electrical conductivity. Here, we have shown that plasma-treated chitin carbon nanotube composite scaffolds show very good neuron adhesion as well as support of synaptic function of neurons. The addition of carbon nanotubes to a chitin biopolymer improved the electrical conductivity and the assisted oxygen plasma treatment introduced more oxygen species onto the chitin nanotube scaffold surface. Neuron viability experiments showed excellent neuron attachment onto plasma-treated chitin nanotube composite scaffolds. The support of synaptic function was evident on chitin/nanotube composites, as confirmed by PSD-95 staining. The biocompatible and electrically-conducting chitin nanotube composite scaffold prepared in this study can be used for in vitro tissue engineering of neurons and, potentially, as an implantable electrode for stimulation and repair of neurons.

The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

PubMed Central

Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

2014-01-01

Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence. PMID:25566225

Mechanical and In Vitro Biological Performance of Graphene Nanoplatelets Reinforced Calcium Silicate Composite

PubMed Central

Mehrali, Mehdi; Moghaddam, Ehsan; Seyed Shirazi, Seyed Farid; Baradaran, Saeid; Mehrali, Mohammad; Latibari, Sara Tahan; Metselaar, Hendrik Simon Cornelis; Kadri, Nahrizul Adib; Zandi, Keivan; Osman, Noor Azuan Abu

2014-01-01

Calcium silicate (CaSiO3, CS) ceramic composites reinforced with graphene nanoplatelets (GNP) were prepared using hot isostatic pressing (HIP) at 1150°C. Quantitative microstructural analysis suggests that GNP play a role in grain size and is responsible for the improved densification. Raman spectroscopy and scanning electron microscopy showed that GNP survived the harsh processing conditions of the selected HIP processing parameters. The uniform distribution of 1 wt.% GNP in the CS matrix, high densification and fine CS grain size help to improve the fracture toughness by ∼130%, hardness by ∼30% and brittleness index by ∼40% as compared to the CS matrix without GNP. The toughening mechanisms, such as crack bridging, pull-out, branching and deflection induced by GNP are observed and discussed. The GNP/CS composites exhibit good apatite-forming ability in the simulated body fluid (SBF). Our results indicate that the addition of GNP decreased pH value in SBF. Effect of addition of GNP on early adhesion and proliferation of human osteoblast cells (hFOB) was measured in vitro. The GNP/CS composites showed good biocompatibility and promoted cell viability and cell proliferation. The results indicated that the cell viability and proliferation are affected by time and concentration of GNP in the CS matrix. PMID:25229540

Targeting αvβ3 and αvβ5 integrins inhibits pulmonary metastasis in an intratibial xenograft osteosarcoma mouse model

PubMed Central

Gvozdenovic, Ana; Boro, Aleksandar; Meier, Daniela; Bode-Lesniewska, Beata; Born, Walter; Muff, Roman; Fuchs, Bruno

2016-01-01

Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvβ3 and αvβ5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvβ3 and αvβ5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvβ5 integrin is mainly found on tumor cells, whereas αvβ3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvβ3 and αvβ5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease. PMID:27409827

Mechanical properties and cytotoxicity of experimental soft lining materials based on urethane acrylate oligomers.

PubMed

Kanie, Takahito; Tomita, Koichi; Tokuda, Masayuki; Arikawa, Hiroyuki; Fujii, Koichi; Ban, Seiji

2009-07-01

The purpose of this investigation was to determine whether experimental light-curing soft lining materials (ESLMs) based on commercially available urethane acrylate oligomers (UA-160TM, UV-3200B, UV-3500BA, and UV-3700B) are suitable for clinical use by measuring their viscosity, compressive modulus, Shore A hardness, tensile strength, adhesive strength, and cytotoxicity. The viscosities of the four ESLMs at 25 degrees C were 10.5 Pa.s, UV-3500BA; 144.0 Pa.s, UA-160TM; 328.8 Pa.s, UV-3700B; and 1079.7 Pa.s, UV-3200B. Polymerized UV-3700B was very soft, whereas the softness of the other ESLMs was similar to that of conventional soft lining materials. No significant difference in adhesive strength was observed between UV-3500BA and UV-3700B at 1 day and those at 12 months. Cytotoxicity was measured by a MTT-based assay using HeLa S3 and Ca9-22 cells. UV-3200B and UV-3700B oligomers and all four polymerized ESLMs showed cell viability over 95.2% (p < 0.05).

Dual laser deposition of Ti:DLC composite for implants

NASA Astrophysics Data System (ADS)

Jelínek, Miroslav; Zemek, Josef; Kocourek, Tomáš; Remsa, Jan; Mikšovský, Jan; Písařík, Petr; Jurek, Karel; Tolde, Zdeněk; Trávníčková, Martina; Vandrovcová, Marta; Filová, Elena

2016-10-01

Ti-doped hydrogen free diamond-like carbon (DLC) layers of dopation up to ~25 at.% were prepared by dual beam pulsed laser deposition (PLD) using two excimer lasers. The arrangement allows continuous fine tuning of dopant concentration on a large scale and deposition flexibility. The layers were prepared on Si(1 0 0) and Ti6Al4V substrates at room temperature. The surface morphology, mechanical properties, bonds, composition, morphology of human osteoblast-like Saos-2 cells, their metabolic activity and production of osteocalcin, a marker of osteogenic cells’ differentiation were tested. The films’ composition changed after x-ray photoelectron spectroscopy (XPS) surface cleaning by argon clusters. Adhesion moved with Ti dopation from 4 N (DLC film) to 11 N (25 at.% of Ti in DLC). Creation of TiC was observed for higher Ti dopation. The contact angle and surface free energy stayed unchanged for higher Ti dopation. Saos-2 cells had the highest metabolic activity/viability on DLC with 10 at.% of Ti and on control polystyrene dishes on days 1 and 3. The Ti dopation improved the formation of vinculin-containing focal adhesion plaques in Saos-2 cells. Immunofluorescence staining revealed similar production of osteocalcin in cells on all tested samples.

Protective Effects of Pistacia lentiscus L. fruit extract against calcium oxalate monohydrate induced proximal tubular injury.

PubMed

Cheraft-Bahloul, Nassima; Husson, Cécile; Ourtioualous, Meriam; Sinaeve, Sébastien; Atmani, Djebbar; Stévigny, Caroline; Nortier, Joëlle L; Antoine, Marie-Hélène

2017-09-14

The world prevalence of kidney stones is increasing and plants are frequently used to treat urolithiasis. Pistacia lentiscus L, a plant which freely grows around the Mediterranean basin areas, is widely used for various pathologies. P. lentiscus has an important impact as it has economical value on top of its pharmacological interest. Decoctions of its aerial parts and/or resin are used to treat kidney stones. To in vitro assess the potential nephroprotective effect of Pistacia lentiscus ethanolic fruit extract (PLEF) on proximal tubular cells in response to the adhesion of calcium oxalate monohydrate (COM) crystals. Human Kidney [HK]-2 cells were incubated with and without COM in the presence or absence of PLEF. Cell viability was measured by the resazurin assay. The expression of E-cadherin was analyzed by PCR. The extracellular production of H 2 O 2 was measured by Amplex® Red H 2 O 2 Assay. The numbers of detached or non-adherent COM crystals in the presence of PLEF were microscopically captured and counted using ImageJ software. The interaction of PLEF with COM and the effect of PLEF on crystal size were analyzed by flow cytometry. The spectrophotometric measurement of turbidity was performed for assessing the COM concentration. PLEF incubated with COM was able to increase the cell viability. The decrease of E-cadherin expression after incubation with COM was counteracted by PLEF. Overproduction of H 2 O 2 induced by COM was also inhibited by PLEF. Observations using flow cytometry showed that interactions between PLEF and the COM crystals occurred. PLEF was also effective in reducing the particles size and in lowering COM concentration. Our data show that COM tubulotoxicity can be significantly reversed by PLEF -at least in part- via an inhibition of COM crystals adhesion onto the apical membrane. This early beneficial effect of PLEF needs to be further investigated as a useful strategy in nephrolithiasis prevention. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Polymer-Ceramic Spiral Structured Scaffolds for Bone Tissue Engineering: Effect of Hydroxyapatite Composition on Human Fetal Osteoblasts

PubMed Central

Zhang, Xiaojun; Chang, Wei; Lee, Paul; Wang, Yuhao; Yang, Min; Li, Jun; Kumbar, Sangamesh G.; Yu, Xiaojun

2014-01-01

For successful bone tissue engineering, a scaffold needs to be osteoconductive, porous, and biodegradable, thus able to support attachment and proliferation of bone cells and guide bone formation. Recently, hydroxyapatites (HA), a major inorganic component of natural bone, and biodegrade polymers have drawn much attention as bone scaffolds. The present study was designed to investigate whether the bone regenerative properties of nano-HA/polycaprolactone (PCL) spiral scaffolds are augmented in an HA dose dependent manner, thereby establishing a suitable composition as a bone formation material. Nano-HA/PCL spiral scaffolds were prepared with different weight ratios of HA and PCL, while porosity was introduced by a modified salt leaching technique. Human fetal osteoblasts (hFOBs) were cultured on the nano-HA/PCL spiral scaffolds up to 14 days. Cellular responses in terms of cell adhesion, viability, proliferation, differentiation, and the expression of bone-related genes were investigated. These scaffolds supported hFOBs adhesion, viability and proliferation. Cell proliferation trend was quite similar on polymer-ceramic and neat polymer spiral scaffolds on days 1, 7, and 14. However, the significantly increased amount of alkaline phosphatase (ALP) activity and mineralized matrix synthesis was evident on the nano-HA/PCL spiral scaffolds. The HA composition in the scaffolds showed a significant effect on ALP and mineralization. Bone phenotypic markers such as bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC), and type I collagen (Col-1) were semi-quantitatively estimated by reverse transcriptase polymerase chain reaction analysis. All of these results suggested the osteoconductive characteristics of HA/PCL nanocomposite and cell maturation were HA dose dependent. For instance, HA∶PCL = 1∶4 group showed significantly higher ALP mineralization and elevated levels of BSP, ON, OC and Col-I expression as compared other lower or higher ceramic ratios. Amongst the different nano-HA/PCL spiral scaffolds, the 1∶4 weight ratio of HA and PCL is shown to be the most optimal composition for bone tissue regeneration. PMID:24475056

Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease.

PubMed

Sivignon, Adeline; de Vallée, Amélie; Barnich, Nicolas; Denizot, Jérémy; Darcha, Claude; Pignède, Georges; Vandekerckove, Pascal; Darfeuille-Michaud, Arlette

2015-02-01

Adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with Crohn's disease (CD), are able to adhere to and invade intestinal epithelial cells. Overexpression of the glycoprotein CEACAM6 on host cells favors AIEC attachment and inflammation. We investigated the ability of Saccharomyces cerevisiae CNCM I-3856 to inhibit AIEC adhesion and to reduce colitis. Adhesion experiments were performed on T84 cells and on enterocytes from patients with CD with AIEC LF82 in the presence of S. cerevisiae. Colonization and symptoms of colitis were assessed in LF82-infected transgenic CEABAC10 mice treated with live S. cerevisiae or S. cerevisiae derivatives. Proinflammatory cytokines were quantified by enzyme linked immunosorbent assay. Intestinal permeability was assessed by measuring the 4 kDa dextran-FITC flux in the serum. S. cerevisiae strongly inhibited LF82 adhesion to T84 cells and to the brush border of CD enterocytes. Yeasts decreased LF82 colonization and colitis in CEABAC10 mice and restored barrier function through prevention of the LF82-induced expression of pore-forming tight junction claudin-2 at the plasma membrane of intestinal epithelial cells. These effects were accompanied by a decrease in proinflammatory cytokines IL-6, IL-1β, and KC release by the gut mucosa. Yeast derivatives exerted similar effects on LF82 colonization and colitis demonstrating that yeast viability was not essential to exert beneficial effects. S. cerevisiae yeasts reduce colitis induced by AIEC bacteria in CEACAM6-expressing mice. Such a probiotic strategy could be envisaged in a subgroup of patients with CD abnormally expressing CEACAM6 at the ileal mucosa and therefore susceptible to being colonized by AIEC bacteria.

Toxicity of a dental adhesive compared with ionizing radiation and zoledronic acid.

PubMed

Alcaraz, Miguel; Olivares, Amparo; Achel, Daniel-Giyngiri; García-Cruz, Emilio; Fondevilla-Soler, Adriana; Canteras-Jordana, Manuel

2015-07-01

To determine the toxicity of aqueous dilutions of a universal self-priming dental adhesive (DA) and comparing these with those elicited by exposure to ionizing radiation (IR), Zoledronic acid (Z) treatment and the synergic effects of the combined treatment with IR+Z. The genotoxic effect of DA was determined by the increase in the frequency of micronuclei in cytokinesis-blocked in cultured human lymphocytes before and after exposure to 2Gy of X-rays. The cytotoxic effect was studied by using the MTT cell viability test in normal prostate cell lines (PNT2) after exposure to different X-ray doses (0Gy-20Gy). The cell lines divided into different groups and treated with different test substances: DA in presence of O2, DA in absence of O2, Z-treated and control. An in vitro dose-dependent and time-dependent cytotoxic effect of DA, Z and IR on PNT2 cells (p>0.001) was demonstrated. DA without-O2, following the recommendations of manufacturers, had a more pronounced effect of increasing cell death than DA with-O2 (p<0.001). In the genotoxicity assay, DA at 25% of its original concentration significantly increased chromosome damage (p<0.001). The samples studied were found to be toxic, and the samples photo-polymerized in absence of O2 showed a bigger cytotoxic effect comparable to the additive toxic effect showed by the combined treatment of IR+Z. Additional effort should be carried out to develop adhesives, which would reduce the release of hazardous substances; since toxic effects are similar to that reported by other agents whose clinical use is controlled by the health authorities.

Biohybrid cochlear implants in human neurosensory restoration.

PubMed

Roemer, Ariane; Köhl, Ulrike; Majdani, Omid; Klöß, Stephan; Falk, Christine; Haumann, Sabine; Lenarz, Thomas; Kral, Andrej; Warnecke, Athanasia

2016-10-07

The success of cochlear implantation may be further improved by minimizing implantation trauma. The physical trauma of implantation and subsequent immunological sequelae can affect residual hearing and the viability of the spiral ganglion. An ideal electrode should therefore decrease post-implantation trauma and provide support to the residual spiral ganglion population. Combining a flexible electrode with cells producing and releasing protective factors could present a potential means to achieve this. Mononuclear cells obtained from bone marrow (BM-MNC) consist of mesenchymal and hematopoietic progenitor cells. They possess the innate capacity to induce repair of traumatized tissue and to modulate immunological reactions. Human bone marrow was obtained from the patients that received treatment with biohybrid electrodes. Autologous mononuclear cells were isolated from bone marrow (BM-MNC) by centrifugation using the Regenlab™ THT-centrifugation tubes. Isolated BM-MNC were characterised using flow cytometry. In addition, the release of cytokines was analysed and their biological effect tested on spiral ganglion neurons isolated from neonatal rats. Fibrin adhesive (Tisseal™) was used for the coating of silicone-based cochlear implant electrode arrays for human use in order to generate biohybrid electrodes. Toxicity of the fibrin adhesive and influence on insertion, as well on the cell coating, was investigated. Furthermore, biohybrid electrodes were implanted in three patients. Human BM-MNC release cytokines, chemokines, and growth factors that exert anti-inflammatory and neuroprotective effects. Using fibrin adhesive as a carrier for BM-MNC, a simple and effective cell coating procedure for cochlear implant electrodes was developed that can be utilised on-site in the operating room for the generation of biohybrid electrodes for intracochlear cell-based drug delivery. A safety study demonstrated the feasibility of autologous progenitor cell transplantation in humans as an adjuvant to cochlear implantation for neurosensory restoration. This is the first report of the use of autologous cell transplantation to the human inner ear. Due to the simplicity of this procedure, we hope to initiate its widespread utilization in various fields.

Knockdown of RhoA expression alters ovarian cancer biological behavior in vitro and in nude mice.

PubMed

Wang, Xiaoxia; Jiang, Wenyan; Kang, Jiali; Liu, Qicai; Nie, Miaoling

2015-08-01

RhoA regulates cell proliferation, migration, angiogenesis and gene expression. Altered RhoA activity contributes to cancer progression. The present study investigated the effects of RhoA knockdown on the regulation of ovarian cancer biological behavior in vitro and in nude mice. The expression of RhoA was knocked down using a lentivirus carrying RhoA short hairpin RNA (shRNA) in ovarian cancer cells and was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The altered ovarian cancer biological behaviors were assayed by cell viability, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL), migration, invasion, and nude mice tumorigenicity assays, while the altered gene expression was detected by RT-qPCR and western blot analysis. The results showed that lentivirus-carrying RhoA shRNA significantly suppressed RhoA expression in ovarian cancer cells, which suppressed tumor cell viability, migration, invasion and adhesion in vitro. RhoA silencing also inhibited the tumorigenicity of ovarian cancer cells in nude mice, which was characterized by the suppression of tumor xenograft formation and growth and induction of tumor cell apoptosis. The results of the present study demonstrated that knockdown of RhoA expression had a significant antitumor effect on ovarian cancer cells in vitro and in nude mice, suggesting that RhoA may be a target for the development of a novel therapeutic strategy in the control of ovarian cancer.

Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

PubMed

Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

2017-03-01

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10 5 ) were infected with T. gondii tachyzoites (5×10 5 ) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10 5 ) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms of T. gondii, giving these drugs new therapeutic uses. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

PubMed

Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S; Tolias, Peter; Yang, Zheng; Lee, Woo Y; Zilberberg, Jenny

2015-01-01

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche

PubMed Central

Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S.; Tolias, Peter; Yang, Zheng

2015-01-01

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC’s viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses. PMID:25973790

Surface Modification of NiTi Alloy via Cathodic Plasma Electrolytic Deposition and its Effect on Ni Ion Release and Osteoblast Behaviors

NASA Astrophysics Data System (ADS)

Yan, Ying; Cai, Kaiyong; Yang, Weihu; Liu, Peng

2013-07-01

To reduce Ni ion release and improve biocompatibility of NiTi alloy, the cathodic plasma electrolytic deposition (CPED) technique was used to fabricate ceramic coating onto a NiTi alloy surface. The formation of a coating with a rough and micro-textured surface was confirmed by X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy, respectively. An inductively coupled plasma mass spectrometry test showed that the formed coating significantly reduced the release of Ni ions from the NiTi alloy in simulated body fluid. The influence of CPED treated NiTi substrates on the biological behaviors of osteoblasts, including cell adhesion, cell viability, and osteogenic differentiation function (alkaline phosphatase), was investigated in vitro. Immunofluorescence staining of nuclei revealed that the CPED treated NiTi alloy was favorable for cell growth. Osteoblasts on CPED modified NiTi alloy showed greater cell viability than those for the native NiTi substrate after 4 and 7 days cultures. More importantly, osteoblasts cultured onto a modified NiTi sample displayed significantly higher differentiation levels of alkaline phosphatase. The results suggested that surface functionalization of NiTi alloy with ceramic coating via the CPED technique was beneficial for cell proliferation and differentiation. The approach presented here is useful for NiTi implants to enhance bone osseointegration and reduce Ni ion release in vitro.

In vitro assessment of stainless steel orthodontic brackets coated with titanium oxide mixed Ag for anti-adherent and antibacterial properties against Streptococcus mutans and Porphyromonas gingivalis.

PubMed

Fatani, Eman Jameel; Almutairi, Hamed H; Alharbi, Ali O; Alnakhli, Yasser Obaidallah; Divakar, Darshan Devang; Muzaheed; Alkheraif, Abdulaziz Abdullah; Khan, Aftab Ahmed

2017-11-01

Orthodontic brackets made from stainless steel were introduced in dentistry, though they have less ability in reducing enamel demineralization and are not successful in preventing microbial as well as biofilm growth. In this study, we evaluated the significant role of different brackets in reducing enamel demineralization indirectly. Results from different tests indicate the significant reduction in adhesion, biofilm formation and slow growth of tested bacterial species on brackets coated with Ag + TiO2 and found to be statistically significant lower than control. There was no loss in cell viability in all brackets indicating that the cells are biocompatible with different bracket materials. Scanning electron microscopy showed less bacteria attached with the surface coated with Ag + TiO2 indicated that bacteria were losing adherent nature on coated surface. In conclusion, TiO2+Ag coating on stainless steel brackets possessed anti-adherent properties and also have demonstrable antibacterial properties therefore helps in preventing dental caries and plaque accumulation indirectly. The cell compatibility of TiO2+Ag coated brackets is superior to the uncoated samples therefore can be used in orthodontics as it not only provide suitable antimicrobial activity and resistance to biofilm formation but also sustained the cell viability of human gingival fibroblast (HGF) cell lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bi-layered collagen nano-structured membrane prototype (collagen matrix 10826(®)) for oral soft tissue regeneration: an "in vitro" study.

PubMed

Nocini, Pier Francesco; Zanotti, Guglielmo; Castellani, Roberto; Grasso, Silvia; Cristofaro, Maria Giulia; De Santis, Daniele

2013-06-01

To evaluate fundamental cell functions, such as adhesion, IL-6 production and proliferation of human gingival keratinocytes cultured on a newly engineered collagen matrix (CM-10826) and to assess the degree of specific biocompatibility of this new device. Primary cultures of human keratinocytes were derived "in vitro" from biopsies of independent donors. Their true epithelial origin was ensured by the expression of cytokeratin 14. Adhesion, proliferation and production of IL-6 cytokine was then measured in the presence or absence of CM-10826 activity or of its relevant components. Functional tests revealed that keratinocytes adhered to CM-10826 and up-regulated their basal IL-6 production. The type of keratinocytes used expressed cytokeratin 14. Proliferation experiments demonstrated that the best cellular response was observed in the presence of Collagen I, the main component of CM-10826. No undesired effects were observed as for keratinocyte viability, morphology or differentiation. Our results demonstrate that CM-10826 has a favourable biological effect on the "in vitro" response of gingival keratinocytes in terms of IL-6 production, cell growth and adhesion. These findings may encourage a possible use of this collagen membrane as a tissue which, alone, may substitute for autologous gingival grafts thereby overcoming the limitations of autologous tissue. © 2012 John Wiley & Sons A/S.

Antibacterial and Antiadhesive Activities of Extracts from Edible Plants against Soft Drink Spoilage by Asaia spp.

PubMed

Antolak, Hubert; Czyzowska, Agata; Kregiel, Dorota

2017-01-01

This study was conducted to investigate the antibacterial and antiadhesive activities of ethanol extracts from five edible plant parts: cinnamon bark ( Cinnamomum zeylanicum ), licorice root ( Glycyrrhiza radix ), nettle leaves ( Urtica dioica ), green tea leaves ( Camellia sinensis ), and elderberry flowers ( Sambucus nigra ). The chemical constituents of the extracts were identified using high-performance liquid chromatography and liquid chromatography plus mass spectrometry. Six strains of Asaia lannensis and Asaia bogorensis bacteria isolated from spoiled commercial fruit-flavored noncarbonated mineral water were used. Bacterial adhesion to polystyrene as an attachment substrate in culture media supplemented with 10% plant extract was evaluated using luminometric measurement of the ATP extracted from adhered cells. The viability of the adhered and planktonic cells was assessed using the plate count method, and the relative adhesion coefficient was calculated. All tested crude extracts contained flavonols (kaempferol, quercetin, and their derivatives), flavanols (catechin and derivatives), flavanones (glabrol, licorice glycoside A, and liquiritin), and phenolic acids (gallic, quinic, chlorogenic, neochlorogenic, caffeic, coumaric, and ferulic). The culture medium with 10% elderberry extract provided the least favorable environment for all tested bacterial strains. Extracts from green tea, cinnamon, and licorice also had significant inhibitory effects on the adhesion of the tested bacterial strains. This research suggests that the addition of selected edible plant extracts could improve the microbial stability of noncarbonated soft drinks.

Biocompatibility and anti-microbiological activity characterization of novel coatings for dental implants: A primer for non-biologists

NASA Astrophysics Data System (ADS)

Monsees, Thomas

2016-08-01

With regard to biocompatibility, the cardinal requirement for dental implants and other medical devices that are in long-term contact with tissue is that the material does not cause any adverse effect to the patient. To warrant stability and function of the implant, proper osseointegration is a further prerequisite. Cells interact with the implant surface as the interface between bulk material and biological tissue. Whereas structuring, deposition of a thin film or other modifications of the surface are crucial parameters in determining favorable adhesion of cells, corrosion of metal surfaces and release of ions can affect cell viability. Both parameters are usually tested using in vitro cytotoxicity and adhesion assays with bone or fibroblasts cells. For bioactive surface modifications, further tests should be considered for biocompatibility evaluation. Depending on the type of modification, this may include analysis of specific cell functions or the determination of antimicrobial activities. The latter is of special importance as bacteria and yeast present in the oral cavity can be introduced during the implantation process and this may lead to chronic infections and implant failure. An antimicrobial coating of the implant is a way to avoid that. This review describes the essential biocompatibility assays for evaluation of new implant materials required by ISO 10993 and also gives an overview on recent test methods for specific coatings of dental implants.

Action of Monomeric/Gemini Surfactants on Free Cells and Biofilm of Asaia lannensis.

PubMed

Koziróg, Anna; Kręgiel, Dorota; Brycki, Bogumił

2017-11-22

We investigated the biological activity of surfactants based on quaternary ammonium compounds: gemini surfactant hexamethylene-1,6-bis-( N,N -dimethyl- N -dodecylammonium bromide) (C6), synthesized by the reaction of N,N -dimethyl- N- dodecylamine with 1,6-dibromohexane, and its monomeric analogue dodecyltrimethylammonium bromide (DTAB). The experiments were performed with bacteria Asaia lannensis , a common spoilage in the beverage industry. The minimal inhibitory concentration (MIC) values were determined using the tube standard two-fold dilution method. The growth and adhesive properties of bacterial cells were studied in different culture media, and the cell viability was evaluated using plate count method. Both of the surfactants were effective against the bacterial strain, but the MIC of gemini compound was significantly lower. Both C6 and DTAB exhibited anti-adhesive abilities. Treatment with surfactants at or below MIC value decreased the number of bacterial cells that were able to form biofilm, however, the gemini surfactant was more effective. The used surfactants were also found to be able to eradicate mature biofilms. After 4 h of treatment with C6 surfactant at concentration 10 MIC, the number of bacterial cells was reduced by 91.8%. The results of this study suggest that the antibacterial activity of the gemini compound could make it an effective microbiocide against the spoilage bacteria Asaia sp. in both planktonic and biofilm stages.

How type 1 fimbriae help Escherichia coli to evade extracellular antibiotics.

PubMed

Avalos Vizcarra, Ima; Hosseini, Vahid; Kollmannsberger, Philip; Meier, Stefanie; Weber, Stefan S; Arnoldini, Markus; Ackermann, Martin; Vogel, Viola

2016-01-05

To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections.

Dynamic monitoring of GPER-mediated estrogenic effects in breast cancer associated fibroblasts: An alternative role of estrogen in mammary carcinoma development.

PubMed

Luo, Haojun; Liu, Manran; Luo, Shujuan; Yu, Tenghua; Wu, Chengyi; Yang, Guanglun; Tu, Gang

2016-08-01

Cancer associated fibroblasts (CAFs) are crucial contributors to breast cancer development. Estrogen affects mammary stroma in both physiological and pathophysiological conditions. We show here that estrogen (G-protein coupled) receptor (GPER) could be detected by immunohistochemistry in stromal fibroblasts of primary breast cancers. The presence of GPER expression was further confirmed by immunofluorescence and quantitative PCR in CAFs isolated from primary breast cancers. Based on dynamic monitoring by real time cell analyzer (RTCA) system, 17-β-estradiol (E2) as well as GPER specific agonist G1 were observed to trigger transient cell index increasing within an hour in a dosage-dependent manner in breast CAFs. In addition, E2 and G1 stimulated intracellular calcium modulation and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 within seconds and minutes in CAFs, respectively. Moreover, E2 and G1 promoted cell proliferation of breast CAFs measured by RTCA monitoring, cell viability assay and cell cycle analysis, and this promotion could be blocked by a GPER-selective antagonist G15. Interestingly, dynamic RTCA monitoring indicated that E2 increased adhesion of resuspended cells, and microscopy confirmed that E2 stimulated cell spreading. Both the adhesion and spreading were proposed to be mediated by GPER, since G1 also stimulated these effects similar to E2, and G15 reduced them. Moreover, GPER was found to mediate migration that was increased by E2 and G1 but reduced by G15 in RTCA cell migration assay and transwell assay. Accordingly, GPER mediates not only rapid actions but also slow effects including adhesion/spreading, proliferation and migration in breast CAFs. Estrogen is likely to affect tumor associated stroma and contributes to mammary carcinoma development through CAFs. Copyright © 2016. Published by Elsevier Inc.

Neutralization of B. anthracis toxins during ex vivo phagocytosis.

PubMed

Tarasenko, Olga; Scott, Ashley; Jones, April; Soderberg, Lee; Alusta, Pierre

2013-07-01

Glycoconjugates (GCs) are recognized as stimulation and signaling agents, affecting cell adhesion, activation, and growth of living organisms. Among GC targets, macrophages are considered ideal since they play a central role in inflammation and immune responses against foreign agents. In this context, we studied the effects of highly selective GCs in neutralizing toxin factors produced by B. anthracis during phagocytosis using murine macrophages. The effects of GCs were studied under three conditions: A) prior to, B) during, and C) following exposure of macrophages to B. anthracis individual toxin (protective antigen [PA], edema factor [EF], lethal factor [LF] or toxin complexes (PA-EF-LF, PA-EF, and PA-LF). We employed ex vivo phagocytosis and post-phagocytosis analysis including direct microscopic observation of macrophage viability, and macrophage activation. Our results demonstrated that macrophages are more prone to adhere to GC-altered PA-EF-LF, PA-EF, and PA-LF toxin complexes. This adhesion results in a higher phagocytosis rate and toxin complex neutralization during phagocytosis. In addition, GCs enhance macrophage viability, activate macrophages, and stimulate nitric oxide (NO) production. The present study may be helpful in identifying GC ligands with toxin-neutralizing and/or immunomodulating properties. In addition, our study could suggest GCs as new targets for existing vaccines and the prospective development of vaccines and immunomodulators used to combat the effects of B. anthracis.

Effect of Bioactive Materials Modified with Chondroitin Sulfate on Human MSC =

NASA Astrophysics Data System (ADS)

De La Torre Torres, Jessica Elizabeth

In this project chondroitin sulfate (CS) and growth factors were studied for their effect on hMSC in biomaterials. First, the effect of these biomolecules was tested in solution. Then, two kinds of biomaterials were created: bioactive surfaces for enhancing bioactivity of implantable devices and bioactive hydrogels which can be used as 3D scaffolds for cell encapsulation and delivery. A pro-survival effect of the growth factors studied in this project (epidermal growth factor, vascular endothelial growth factor and fibroblast growth factor) was not observed when tested in solution, therefore the project further focused on CS effect only. Interestingly, CS did not affect cell growth in media containing serum, while inducing cell detachment from substrate in serum free conditions. For the bioactive surfaces construction, CS was grafted to either an amine-rich plasmapolymerized coating created on polyethylene terephthalate (PET) films (further referred as LP) or to commercial cell culture plates functionalized with amino groups. The bioactive surfaces were characterized by different techniques such as contact angle, atomic force microscopy, Orange II dye and Toluidine Blue O dye colorimetric assays (for amino group and CS quantification respectively) and finally, cell culture experiments (adhesion, growth and survival). Results confirmed the presence of CS grafted on both substrates. Commercial amine plates grafted almost five times more CS compared to LP. This rendered the surface antifouling for proteins and cells as confirmed by protein adsorption and cell culture assays. Cell culture assays on bioactive surfaces based on LP demonstrated improved cell adhesion and growth when compared to tissue culture plates or bare PET films in serum containing conditions. Chitosan based hydrogels containing CS at a concentration of 500 mug/ml resulted in a cohesive hydrogel which supported hMSC viability up to 7 days. However increasing CS concentration to high level such as 10000 mug/ml led to decrease of cell viability after 4 or 7 days, probably due to lack of porosity and water since the hydrogel precipitates upon formation and expulses water. In conclusion, this work demonstrated that CS immobilization can enhance the biological interactions of hMSC with biomaterials used for implantable devices such as PET. Further studies are needed to evaluate the possible effect of CS on hMSC differentiation and phenotype. Hydrogels with CS could be very interesting for tissue engineering applications such as cartilage formation. (Abstract shortened by ProQuest.).

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments.

PubMed

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Yan; McGraw, John; Woods, Matthew; Murray, Stuart; Eckert, Amy; Liu, Wei; Ryan, Terence E

2011-06-01

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.

A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroin-alginate microcarriers using light sheet fluorescent microscopy

PubMed Central

Pierini, Michela; Bevilacqua, Alessandro; Torre, Maria Luisa; Lucarelli, Enrico

2017-01-01

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading. In this work, we propose a non-invasive and non-destructive imaging analysis for investigating motility, viability and distribution of Mesenchymal Stem Cells (MSCs) on silk fibroin-based alginate microcarriers, to test the adhesion capacity of the fibroin coating onto alginate which is known to be unsuitable for cell adhesion. However, in depth characterization of the biomaterial is beyond the scope of this paper. Scaffold-loaded MSCs were stained with Calcein-AM and Ethidium homodimer-1 to detect live and dead cells, respectively, and counterstained with Hoechst to label cell nuclei. Time-lapse Light Sheet Fluorescent Microscopy (LSFM) was then used to produce three-dimensional images of the entire cells-loaded fibroin/alginate microcarriers. In order to quantitatively track the cell motility over time, we also developed an open source user friendly software tool called Fluorescent Cell Tracker in Three-Dimensions (F-Tracker3D). Combining LSFM with F-Tracker3D we were able for the first time to assess the distribution and motility of stem cells in a non-invasive, non-destructive, quantitative, and three-dimensional analysis of the entire surface of the cell-loaded scaffold. We therefore propose this imaging technique as an innovative holistic tool for monitoring cell-biomaterial interactions, and as a tool for the design, fabrication and functionalization of a scaffold as a medical device. PMID:28817694

Effects of Long-Term 50Hz Power-Line Frequency Electromagnetic Field on Cell Behavior in Balb/c 3T3 Cells

PubMed Central

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn’t change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein. PMID:25695503

Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

PubMed

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn't change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein.

Production, characterisation, and cytocompatibility of porous titanium-based particulate scaffolds.

PubMed

Luthringer, B J C; Ali, F; Akaichi, H; Feyerabend, F; Ebel, T; Willumeit, R

2013-10-01

Despite its non-matching mechanical properties titanium remains the preferred metal implant material in orthopaedics. As a consequence in some cases stress shielding effect occurs, leading to implant loosening, osteopenia, and finally revision surgery. Porous metal scaffolds to allow easier specialised cells ingrowth with mechanical properties closer to the ones of bone can overcome this problem. This should improve healing processes, implant integration, and dynamic strength of implants retaining. Three Ti-6Al-4V materials were metal injection moulded and tailored porosities were effectively achieved. After microstructural and mechanical characterisation, two different primary cells of mesenchymal origin (human umbilical cord perivascular cells and human bone derived cells which revealed to be two pertinent models) as well as one cell line originated from primary osteogenic sarcoma, Saos-2, were bestowed to investigate cell-material interaction on genomic and proteome levels. Biological examinations disclosed that no material has negative impact on early adhesion, proliferation or cell viability. An efficient cell ingrowth into material with an average porosity of 25-50 μm was proved.

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

PubMed Central

Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

2012-01-01

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

Investigation of welded interconnection of large area wraparound contacted silicon solar cells

NASA Technical Reports Server (NTRS)

Lott, D. R.

1984-01-01

An investigation was conducted to evaluate the welding and temperature cycle testing of large area 5.9 x 5.9 wraparound silicon solar cells utilizing printed circuit substrates with SSC-155 interconnect copper metals and the LMSC Infrared Controlled weld station. An initial group of 5 welded modules containing Phase 2 developmental 5.9 x 5.9 cm cells were subjected to cyclical temperatures of + or 80 C at a rate of 120 cycles per day. Anomalies were noted in the adhesion of the cell contact metallization; therefore, 5 additional modules were fabricated and tested using available Phase I cells with demonstrated contact integrity. Cycling of the later module type through 12,000 cycles indicated the viability of this type of lightweight flexible array concept. This project demonstrated acceptable use of an alternate interconnect copper in combination with large area wraparound cells and emphasized the necessity to implement weld pull as opposed to solder pull procedures at the cell vendors for cells that will be interconnected by welding.

The effects of sulforaphane on canine osteosarcoma proliferation and invasion.

PubMed

Rizzo, V L; Levine, C B; Wakshlag, J J

2017-09-01

Recent evidence in in vitro and in vivo models suggests that sulforaphane (SFN), found in raw cruciferous vegetables, may have utility in chemoprevention, as an antineoplastic agent and as a free radical scavenger. The effects of SFN alone or with doxorubicin on cell viability were examined, as well as cell cycle kinetics, invasion capabilities and apoptosis in three canine osteosarcoma cell line (D17, OS 2.4 and HMPOS). Results showed that SFN could not induce cell death at potentially physiological concentrations (<50 μM), but significantly diminished cell invasion and downregulation of focal adhesion kinase (FAK) signaling. Modest cell cycle changes were observed in each cell line. When doxorubicin was used in conjunction with SFN, there was a protective effect to doxorubicin-induced cytotoxicity in D17 and OS 2.4 cells. Further studies examining SFN as a supplement are warranted, particularly in light of pro-proliferative and cytoprotective properties in canine osteosarcoma. © 2016 John Wiley & Sons Ltd.

Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

PubMed

Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

2015-01-01

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines.

PubMed

Heindl, Stefan; Eggenstein, Evelyn; Keller, Simone; Kneissl, Julia; Keller, Gisela; Mutze, Kathrin; Rauser, Sandra; Gasteiger, Georg; Drexler, Ingo; Hapfelmeier, Alexander; Höfler, Heinz; Luber, Birgit

2012-05-01

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line. EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance. These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

PubMed

Patrulea, V; Hirt-Burri, N; Jeannerat, A; Applegate, L A; Ostafe, V; Jordan, O; Borchard, G

2016-05-20

RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

The influences of N-acetyl cysteine (NAC) on the cytotoxicity and mechanical properties of Poly-methylmethacrylate (PMMA)-based dental resin

PubMed Central

Jiao, Yang; Ma, Sai; Li, Jing; Shan, Lequn; Yang, Yanwei; Li, Meng

2015-01-01

Objectives. This study aimed to investigate the influences of N-acetyl cysteine (NAC) on cytotoxicity and mechanical properties of Poly-methylmethacrylate (PMMA) dental resins. Methods. Experimental PMMA resin was prepared by incorporating various concentrations of NAC (0, 0.15, 0.3, 0.6 and 0.9 wt.%). MTT assay was performed to investigate viability of human dental pulp cells after exposure to extract of PMMA resin with or without NAC. Cell adhesion on resin specimens was examined with scanning electron microscopy. Degree of conversion was studied with Fourier Transform Infrared Spectroscopy (FTIR). Flexural strength, microhardness and surface roughness was evaluated using a universal testing machine, microhardness tester and optical profilometer, respectively. Results. Incorporation of NAC into PMMA resin significantly reduced its cytotoxicity and enhanced cell adhesion on its surface. NAC induced negative influences on the mechanical and physical properties of PMMA resin in a dose-dependent manner. The degree of conversion for all experimental PMMA resins reached as high as 72% after 24 h of polymerization. All the tested properties were maintained when the concentration of incorporated NAC was 0.15 wt.%. Conclusion. The addition of 0.15 wt.% NAC remarkably improved biocompatibility of PMMA resin without exerting significant negative influence on its mechanical and physical properties. PMID:25922788

Synthesis of poly(ester-carbonate) with a pendant acetylcholine analog for promoting neurite growth.

PubMed

Xing, Dongming; Ma, Lie; Gao, Changyou

2014-10-01

The modification of biodegradable polyesters with bioactive molecules has become an important strategy for controlling neuron adhesion and neurite outgrowth in nerve regeneration. In this study we report a biodegradable poly(ester-carbonate) with a pendant acetylcholine analog, which a neurotransmitter for the enhancement of neuron adhesion and outgrowth. The acetylcholine-functionalized poly(ester-carbonate) (Ach-P(LA-ClTMC)) was prepared by copolymerizing l-lactide (LA) and 5-methyl-5-chloroethoxycarbonyl trimethylene carbonate (ClTMC), followed by quaternization with trimethylamine. The acetylcholine analog content could be modulated by changing the molar feeding fraction of ClTMC. The incorporation of the acetylcholine analog improved the hydrophilicity of the films, but the acetylcholine analog content did not significantly influence the surface morphology of the acetylcholine-functionalized films. The results of PC12 cell culture showed that the acetylcholine analog promoted cell viability and neurite outgrowth in a concentration-dependent manner. The longest length of neurite and the percentage of cells bearing neurites were obtained on the Ach-P(LA-ClTMC)-10 film. All the results indicate that the integration of the acetylcholine analog at an appropriate fraction could be an effective strategy for optimizing the existing biodegradable polyesters for nerve regeneration applications. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs.

PubMed

Silva, Karina R; Rezende, Rodrigo A; Pereira, Frederico D A S; Gruber, Peter; Stuart, Mellannie P; Ovsianikov, Aleksandr; Brakke, Ken; Kasyanov, Vladimir; da Silva, Jorge V L; Granjeiro, José M; Baptista, Leandra S; Mironov, Vladimir

2016-01-01

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young's modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

Biofunctionalization of a titanium surface with a nano-sawtooth structure regulates the behavior of rat bone marrow mesenchymal stem cells

PubMed Central

Zhang, Wenjie; Li, Zihui; Liu, Yan; Ye, Dongxia; Li, Jinhua; Xu, Lianyi; Wei, Bin; Zhang, Xiuli; Liu, Xuanyong; Jiang, Xinquan

2012-01-01

Background: The topography of an implant surface can serve as a powerful signaling cue for attached cells and can enhance the quality of osseointegration. A series of improved implant surfaces functionalized with nanoscale structures have been fabricated using various methods. Methods: In this study, using an H2O2 process, we fabricated two size-controllable sawtooth-like nanostructures with different dimensions on a titanium surface. The effects of the two nano-sawtooth structures on rat bone marrow mesenchymal stem cells (BMMSCs) were evaluated without the addition of osteoinductive chemical factors. Results: These new surface modifications did not adversely affect cell viability, and rat BMMSCs demonstrated a greater increase in proliferation ability on the surfaces of the nano-sawtooth structures than on a control plate. Furthermore, upregulated expression of osteogenic-related genes and proteins indicated that the nano-sawtooth structures promote osteoblastic differentiation of rat BMMSCs. Importantly, the large nano-sawtooth structure resulted in the greatest cell responses, including increased adhesion, proliferation, and differentiation. Conclusion: The enhanced adhesion, proliferation, and osteogenic differentiation abilities of rat BMMSCs on the nano-sawtooth structures suggest the potential to induce improvements in bone-titanium integration in vivo. Our study reveals the key role played by the nano-sawtooth structures on a titanium surface for the fate of rat BMMSCs and provides insights into the study of stem cell-nanostructure relationships and the related design of improved biomedical implant surfaces. PMID:22927760

Streptococcus mitis/human gingival fibroblasts co-culture: the best natural association in answer to the 2-hydroxyethyl methacrylate release

PubMed Central

Di Giulio, Mara; D'Ercole, Simonetta; Zara, Susi; Cataldi, Amelia; Cellini, Luigina

2012-01-01

One of the major components of dental polymerized resin-based restorative materials is 2-hydroxyethyl methacrylate (HEMA) and its release in monomeric form interferes with the oral cavity environment. This study aimed to evaluate HEMA monomeric effects on the co-culture of Streptococcus mitis and human gingival fibroblasts (HGFs). Streptococcus mitis DS12 and S. mitis ATCC 6249 were co-cultivated with HGF in the presence of HEMA (3 mM), for 48 and 72 h; the amount of sessile and planktonic cells, as well as the prokaryotic and eukaryotic cell viability were analyzed in treated and untreated samples. The treatment of S. mitis/HGFs with HEMA did not produce significant effects on the bacterial adhesion and induced an increase in planktonic S. mitis ATCC 6249 population after 48 and 72 h. HEMA increased significantly the planktonic S. mitis ATCC 6249 viability when co-cultured with HGFs, while a cytotoxic effect on HGFs, without bacteria, was recorded. An increase of bacterial aggregation on HGFs was also detected with HEMA. Data obtained in this study suggest that HEMA exhibits a toxic effect mainly on eukaryotic cells and this effect can be modulated by co-cultivation with the S. mitis cells which, in the presence of the monomer, enhance their aggregation rate on HGFs. PMID:22229269

Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction

PubMed Central

Scioli, Maria Giovanna; Lo Giudice, Pietro; Bielli, Alessandra; Tarallo, Valeria; De Rosa, Alfonso; De Falco, Sandro; Orlandi, Augusto

2015-01-01

Background Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery. Methods and Results We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction. Conclusion PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers. PMID:26473356

S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

PubMed

Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

2016-11-01

Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells.

PubMed

Nair, Manitha B; Bernhardt, Anne; Lode, Anja; Heinemann, Christiane; Thieme, Sebastian; Hanke, Thomas; Varma, Harikrishna; Gelinsky, Michael; John, Annie

2009-08-01

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.

Photoelectrical Stimulation of Neuronal Cells by an Organic Semiconductor-Electrolyte Interface.

PubMed

Abdullaeva, Oliya S; Schulz, Matthias; Balzer, Frank; Parisi, Jürgen; Lützen, Arne; Dedek, Karin; Schiek, Manuela

2016-08-23

As a step toward the realization of neuroprosthetics for vision restoration, we follow an electrophysiological patch-clamp approach to study the fundamental photoelectrical stimulation mechanism of neuronal model cells by an organic semiconductor-electrolyte interface. Our photoactive layer consisting of an anilino-squaraine donor blended with a fullerene acceptor is supporting the growth of the neuronal model cell line (N2A cells) without an adhesion layer on it and is not impairing cell viability. The transient photocurrent signal upon illumination from the semiconductor-electrolyte layer is able to trigger a passive response of the neuronal cells under physiological conditions via a capacitive coupling mechanism. We study the dynamics of the capacitive transmembrane currents by patch-clamp recordings and compare them to the dynamics of the photocurrent signal and its spectral responsivity. Furthermore, we characterize the morphology of the semiconductor-electrolyte interface by atomic force microscopy and study the stability of the interface in dark and under illuminated conditions.

In vitro proliferation and osteogenic differentiation of mesenchymal stem cells on nanoporous alumina

PubMed Central

Song, Yuanhui; Ju, Yang; Song, Guanbin; Morita, Yasuyuki

2013-01-01

Cell adhesion, migration, and proliferation are significantly affected by the surface topography of the substrates on which the cells are cultured. Alumina is one of the most popular implant materials used in orthopedics, but few data are available concerning the cellular responses of mesenchymal stem cells (MSCs) grown on nanoporous structures. MSCs were cultured on smooth alumina substrates and nanoporous alumina substrates to investigate the interaction between surface topographies of nanoporous alumina and cellular behavior. Nanoporous alumina substrates with pore sizes of 20 nm and 100 nm were used to evaluate the effect of pore size on MSCs as measured by proliferation, morphology, expression of integrin β1, and osteogenic differentiation. An MTT assay was used to measure cell viability of MSCs on different substrates, and determined that cell viability decreased with increasing pore size. Scanning electron microscopy was used to investigate the effect of pore size on cell morphology. Extremely elongated cells and prominent cell membrane protrusions were observed in cells cultured on alumina with the larger pore size. The expression of integrin β1 was enhanced in MSCs cultured on porous alumina, revealing that porous alumina substrates were more favorable for cell growth than smooth alumina substrates. Higher levels of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin, and mineralization were detected in cells cultured on alumina with 100 nm pores compared with cells cultured on alumina with either 20 nm pores or smooth alumina. This work demonstrates that cellular behavior is affected by variation in pore size, providing new insight into the potential application of this novel biocompatible material for the developing field of tissue engineering. PMID:23935364

Amoebicidal activity of curcumin on Entamoeba histolytica trophozoites.

PubMed

Rangel-Castañeda, Itzia Azucena; Hernández-Hernández, José Manuel; Pérez-Rangel, Armando; González-Pozos, Sirenia; Carranza-Rosales, Pilar; Charles-Niño, Claudia Lisette; Tapia-Pastrana, Gabriela; Ramírez-Herrera, Mario Alberto; Castillo-Romero, Araceli

2018-03-01

This study was undertaken to investigate the amoebicidal potential of curcumin on Entamoeba histolytica, as well as its synergistic effect with metronidazole. Entamoeba histolytica trophozoites were exposed to 100, 200 and 300 μm of curcumin, for 6, 12 and 24 h. Consequently, the viability of cells was determined by trypan blue exclusion test. All specimens were further analysed by scanning electron microscopy. For drug combination experiment, the Chou-Talalay method was used. Curcumin affected the growth and cell viability in a time- and dose-dependent manner. The higher inhibitory effects were observed with 300 μm at 24 h; 65.5% of growth inhibition and only 28.8% of trophozoites were viable. Additionally, curcumin also altered adhesion and the morphology of the trophozoites. Scanning electron microscopy revealed treated trophozoites with damages on the membrane, size alterations and parasites with loss of cellular integrity. In addition, the combination of curcumin + metronidazole exhibited a synergistic effect; the activity of both drugs was improved. This is the first report evaluating the effectiveness of curcumin against E. histolytica. Our results suggest that CUR could be considered for evaluation in future pharmacological studies as a promising amoebicidal agent or as complementary therapy. © 2018 Royal Pharmaceutical Society.

L-histidine inhibits biofilm formation and FLO11-associated phenotypes in Saccharomyces cerevisiae flor yeasts.

PubMed

Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

2014-01-01

Flor yeasts of Saccharomyces cerevisiae have an innate diversity of Flo11p which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling Flo11p alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce Flo11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to Flo11p expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air-liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the Flo11p gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts [corrected].

L-Histidine Inhibits Biofilm Formation and FLO11-Associated Phenotypes in Saccharomyces cerevisiae Flor Yeasts

PubMed Central

Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

2014-01-01

Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to FLO11 expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air–liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the FLO11 gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts. PMID:25369456

Developing a tissue-engineered neural-electrical relay using encapsulated neuronal constructs on conducting polymer fibers.

PubMed

Cullen, D Kacy; R Patel, Ankur; Doorish, John F; Smith, Douglas H; Pfister, Bryan J

2008-12-01

Neural-electrical interface platforms are being developed to extracellularly monitor neuronal population activity. Polyaniline-based electrically conducting polymer fibers are attractive substrates for sustained functional interfaces with neurons due to their flexibility, tailored geometry and controlled electro-conductive properties. In this study, we addressed the neurobiological considerations of utilizing small diameter (<400 microm) fibers consisting of a blend of electrically conductive polyaniline and polypropylene (PA-PP) as the backbone of encapsulated tissue-engineered neural-electrical relays. We devised new approaches to promote survival, adhesion and neurite outgrowth of primary dorsal root ganglion neurons on PA-PP fibers. We attained a greater than ten-fold increase in the density of viable neurons on fiber surfaces to approximately 700 neurons mm(-2) by manipulating surrounding surface charges to bias settling neuronal suspensions toward fibers coated with cell-adhesive ligands. This stark increase in neuronal density resulted in robust neuritic extension and network formation directly along the fibers. Additionally, we encapsulated these neuronal networks on PA-PP fibers using agarose to form a protective barrier while potentially facilitating network stability. Following encapsulation, the neuronal networks maintained integrity, high viability (>85%) and intimate adhesion to PA-PP fibers. These efforts accomplished key prerequisites for the establishment of functional electrical interfaces with neuronal populations using small diameter PA-PP fibers-specifically, improved neurocompatibility, high-density neuronal adhesion and neuritic network development directly on fiber surfaces.

Enhanced growth and osteogenic differentiation of human osteoblast-like cells on boron-doped nanocrystalline diamond thin films.

PubMed

Grausova, Lubica; Kromka, Alexander; Burdikova, Zuzana; Eckhardt, Adam; Rezek, Bohuslav; Vacik, Jiri; Haenen, Ken; Lisa, Vera; Bacakova, Lucie

2011-01-01

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH(4):H(2) gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000 ± 14,000 and 152,000 ± 10,000 cells/cm(2), respectively, compared to 113,000 ± 10,000 cells/cm(2) on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells.

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

PubMed Central

Grausova, Lubica; Kromka, Alexander; Burdikova, Zuzana; Eckhardt, Adam; Rezek, Bohuslav; Vacik, Jiri; Haenen, Ken; Lisa, Vera; Bacakova, Lucie

2011-01-01

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells. PMID:21695172

Protective potential of non-dialyzable material fraction of cranberry juice on the virulence of P. gingivalis and F. nucleatum mixed infection.

PubMed

Polak, David; Naddaf, Raja; Shapira, Lior; Weiss, Ervin I; Houri-Haddad, Yael

2013-07-01

Periodontitis is a polymicrobial infectious disease. A novel potential chemical treatment modality may lie in bacterial anti-adhesive materials, such as cranberry juice fractions. The aim of this study is to explore the effect of high molecular weight cranberry constituent (non-dialyzable material [NDM]) on the virulence of a mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in mice. In vitro, the anti-adhesive property of NDM was validated on epithelial cell culture, and inhibition of coaggregation was tested using a coaggregation assay. The in vivo effect was tested on the outcome of experimental periodontitis induced by a P. gingivalis and F. nucleatum mixed infection, and also on the local host response using the subcutaneous chamber model of infection. Phagocytosis was also tested on RAW macrophages by the use of fluorescent-labeled bacteria. NDM was found to inhibit the adhesion of both species of bacteria onto epithelial cells and to inhibit coaggregation in a dose-dependent manner. NDM consumption by mice attenuated the severity of experimental periodontitis compared with a mixed infection without NDM treatment. In infected subcutaneous chambers, NDM alone reduced tumor necrosis factor-α (TNF-α) levels induced by the mixed infection. In vitro, NDM eliminated TNF-α expression by macrophages that were exposed to P. gingivalis and F. nucleatum, without impairing their viability. Furthermore, NDM increased the phagocytosis of P. gingivalis. The results indicate that the use of NDM may hold potential protective and/or preventive modalities in periodontal disease. Underlying mechanisms for this trait may perhaps be the anti-adhesive properties of NDM or its potential effect on inflammation.

Anti-adhesive and pro-apoptotic effects of 2-hydroxyethyl methacrylate on human gingival fibroblasts co-cultured with Streptococcus mitis strains

PubMed Central

Zara, S; Di Giulio, M; D’Ercole, S; Cellini, L; Cataldi, A

2011-01-01

Aim To evaluate and observe the cellular reactions that occur during the interaction/integration between 2-hydroxyethyl methacrylate/host tissue/microbial environment, in a co-culture of human gingival fibroblasts (HGF) and Streptococcus mitis strains. Methodology Streptococcus mitis were cultured with strains in the presence of 3 mmol L−1 HEMA for 48 h and 72 h. Cytotoxicity was evaluated by the trypan blue dye exclusion test. Apoptosis was evaluated by TUNEL analysis. Adhesion was evaluated by immunofluorescence and western blot analyses. Quantitative analyses of the results were acquired by Qwin Plus 3.5 and QuantityOne I-D analysis software, respectively. The statistical significance of the results was evaluated using t-tests and linear regression tests. Results The trypan blue dye test revealed 47.3% and 46.5% of dead fibroblasts after 48 and 72 h HEMA treatment, respectively, while bacterial viability was not influenced by the presence of HEMA and fibroblasts. The expression of pro-collagen I, involved in fibroblast adhesion, in untreated samples ranged from 12.49% to 6.91% of the positive area after 48 and 72 h, respectively, dropping to below 2% of the positive area in the other experimental conditions. Unlike the trypan blue test, co-cultured samples treated with HEMA showed 20% and 25% versus 17% and 21% (after 48 and 72 h, respectively) of apoptotic cells. Conclusions The evidence for HEMA toxicity and anti-adhesive effects against eukaryotic cells was reduced in the presence of bacteria, suggesting that dental resins should be well polymerized to avoid the spread of toxic monomers within the mouth. PMID:21902700

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications.

PubMed

Ribeiro, A R; Oliveira, F; Boldrini, L C; Leite, P E; Falagan-Lotsch, P; Linhares, A B R; Zambuzzi, W F; Fragneaud, B; Campos, A P C; Gouvêa, C P; Archanjo, B S; Achete, C A; Marcantonio, E; Rocha, L A; Granjeiro, J M

2015-09-01

Titanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration. Copyright © 2015 Elsevier B.V. All rights reserved.

How type 1 fimbriae help Escherichia coli to evade extracellular antibiotics

PubMed Central

Avalos Vizcarra, Ima; Hosseini, Vahid; Kollmannsberger, Philip; Meier, Stefanie; Weber, Stefan S.; Arnoldini, Markus; Ackermann, Martin; Vogel, Viola

2016-01-01

To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections. PMID:26728082

Chemical modification of polyvinyl chloride and silicone elastomer in inhibiting adhesion of Aeromonas hydrophila.

PubMed

Kregiel, Dorota; Berlowska, Joanna; Mizerska, Urszula; Fortuniak, Witold; Chojnowski, Julian; Ambroziak, Wojciech

2013-07-01

Disease-causing bacteria of the genus Aeromonas are able to adhere to pipe materials, colonizing the surfaces and forming biofilms in water distribution systems. The aim of our research was to study how the modification of materials used commonly in the water industry can reduce bacterial cell attachment. Polyvinyl chloride and silicone elastomer surfaces were activated and modified with reactive organo-silanes by coupling or co-crosslinking silanes with the native material. Both the native and modified surfaces were tested using the bacterial strain Aeromonas hydrophila, which was isolated from the Polish water distribution system. The surface tension of both the native and modified surfaces was measured. To determine cell viability and bacterial adhesion two methods were used, namely plate count and luminometry. Results were expressed in colony-forming units (c.f.u.) and in relative light units (RLU) per cm(2). Almost all the chemically modified surfaces exhibited higher anti-adhesive and anti-microbial properties in comparison to the native surfaces. Among the modifying agents examined, poly[dimethylsiloxane-co-(N,N-dimethyl-N-n-octylammoniopropyl chloride) methylsiloxane)] terminated with hydroxydimethylsilyl groups (20 %) in silicone elastomer gave the most desirable results. The surface tension of this modifier, was comparable to the non-polar native surface. However, almost half of this value was due to the result of polar forces. In this case, in an adhesion analysis, only 1 RLU cm(-2) and less than 1 c.f.u. cm(-2) were noted. For the native gumosil, the results were 9,375 RLU cm(-2) and 2.5 × 10(8) c.f.u. cm(-2), respectively. The antibacterial activity of active organo-silanes was associated only with the carrier surface because no antibacterial compounds were detected in liquid culture media, in concentrations that were able to inhibit cell growth.

Galectin-3 alters the lateral mobility and clustering of β1-integrin receptors

PubMed Central

Yang, Esther H.; Rode, Julia; Howlader, Md. Amran; Eckermann, Marina; Santos, Jobette T.; Hernandez Armada, Daniel; Zheng, Ruixiang; Zou, Chunxia

2017-01-01

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5β1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased β1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3–integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins. PMID:29016609

Development of a simple and convenient cell-based electrochemical biosensor for evaluating the individual and combined toxicity of DON, ZEN, and AFB1.

PubMed

Xia, Shuang; Zhu, Pei; Pi, Fuwei; Zhang, Yinzhi; Li, Yun; Wang, Jiasheng; Sun, Xiulan

2017-11-15

A simple and convenient cell-based electrochemical biosensor was developed to assess the individual and combined toxicity of deoxynivalenol (DON), zearalenone (ZEN), and Aflatoxin B 1 (AFB 1 ) on Hep G2 cells. The sensor was modified in succession with AuNPs (gold nanoparticles), cysteamine, and laminin. The cells interacting with laminin formed tight cell-to-electrode contacts, and collagen was used to maintain cell adhesion and viability. Electrochemical impedance spectroscopy (EIS) was developed to evaluate mycotoxin toxicity. Experimental results show that DON, ZEN, and AFB 1 caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of DON, ZEN, and AFB 1 in the range of 0.01-20, 0.1-50, and 0.1-3.5μg/mL, and IC 50 obtained using the developed method was 48.5, 59.0, and 3.10μg/mL, respectively. A synergistic effect was observed between DON and ZEN, an additive effect was observed between DON and AFB 1 , and an antagonism effect was found in the binary mixtures of ZEN and AFB 1 and ternary mixtures. These results were confirmed via CCK-8 assay. Utilizing SEM, we found that cells treated with mycotoxins caused significant changes in cell morphology, thus lessening cell adsorption and impedance reduction. Biological assay indicated that EIS patterns correlated with [Ca 2+ ] i concentrations and apoptosis and necrotic cells ratios, thus effecting electrochemical signals. This method is simpler, more convenient, sensitive, and has a quicker response rate than most conventional cytotoxicity evaluation methods. Copyright © 2017. Published by Elsevier B.V.

Overexpression of HepaCAM inhibits cell viability and motility through suppressing nucleus translocation of androgen receptor and ERK signaling in prostate cancer.

PubMed

Song, Xuedong; Wang, Yin; Du, Hongfei; Fan, Yanru; Yang, Xue; Wang, Xiaorong; Wu, Xiaohou; Luo, Chunli

2014-07-01

HepaCAM is suppressed in a variety of human cancers, and involved in cell adhesion, growth, migration, invasion, and survival. However, the expression and function of HepaCAM in prostate cancer are still unknown. HepaCAM expression has been detected by RT-PCR, Western blotting and immunohistochemistry staining in prostate cell lines RWPE-1, LNCap, DU145, PC3, and in 75 human prostate tissue specimens, respectively. Meanwhile, the cell proliferation ability was detected by WST-8 assay. The role of HepaCAM in prostate cancer cell migration and invasion was examined by wound healing and transwell assay. And flow cytometry was used to observe the apoptosis of prostate cancer cells. Then we detected changes of Androgen Receptor translocation and ERK signaling using immunofluorescence staining and western blot after overexpression of HepaCAM. The HepaCAM expression was significantly down-regulated in prostate cancer tissues and undetected in prostate cancer cells. However, the low HepaCAM expression was not statistically associated with clinicopathological characteristics of prostate cancer. Overexpression of HepaCAM in prostate cancer cells decreased the cell proliferation, migration and invasion, and induced the cell apoptosis. Meanwhile, HepaCAM prevented the androgen receptor translocation from the cytoplasm to the nucleus and down-regulated the MAPK/ERK signaling. Our results suggested that HepaCAM acted as a tumor suppressor in prostate cancer. HepaCAM inhibited cell viability and motility which might be through suppressing the nuclear translocation of Androgen Receptor and down-regulating the ERK signaling. Therefore, it was indicated that HepaCAM may be a potential therapeutic target for prostate cancer. © 2014 Wiley Periodicals, Inc.

GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation and growth factor production for enhanced tumor cell proliferation

PubMed Central

Curran, Colleen S.; Evans, Michael D.; Bertics, Paul J.

2011-01-01

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, human primary blood eosinophils were cultured with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results revealed differential eosinophil adhesion and increased survival in response to co-culture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression and S100A9 release. Addition of GM-CSF neutralizing antibodies to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF producing tumors and eosinophil-derived growth factors in tumor promotion/progression. PMID:21705618

Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells.

PubMed

Yang, Po-Min; Wu, Zhi-Zhen; Zhang, Yu-Qi; Wung, Being-Sun

2016-06-15

Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2- induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

PubMed

Tresoldi, Claudia; Stefani, Ilaria; Ferracci, Gaia; Bertoldi, Serena; Pellegata, Alessandro F; Farè, Silvia; Mantero, Sara

2017-04-26

In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

Composite vascular scaffold combining electrospun fibers and physically-crosslinked hydrogel with copper wire-induced grooves structure.

PubMed

Liu, Yuanyuan; Jiang, Chen; Li, Shuai; Hu, Qingxi

2016-08-01

While the field of tissue engineered vascular grafts has greatly advanced, many inadequacies still exist. Successfully developed scaffolds require mechanical and structural properties that match native vessels and optimal microenvironments that foster cell integration, adhesion and growth. We have developed a small diameter, three-layered composite vascular scaffold which consists of electrospun fibers and physically-crosslinked hydrogel with copper wire-induced grooves by combining the electrospinning and dip-coating methods. Scaffold morphology and mechanics were assessed, quantified and compared to native vessels. Scaffolds were seeded with Human Umbilical Vein Endothelial Cells (HUVECs), cultured in vitro for 3 days and were evaluated for cell viability and morphology. The results showed that composite scaffolds had adjustable mechanical strength and favorable biocompatibility, which is important in the future clinical application of Tissue-engineered vascular grafts (TEVGs). Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate cancer in vitro. Potential effect against MMP-9.

PubMed

Lobo, Gricela; Monasterios, Melina; Rodrigues, Juan; Gamboa, Neira; Capparelli, Mario V; Martínez-Cuevas, Javier; Lein, Michael; Jung, Klaus; Abramjuk, Claudia; Charris, Jaime

2015-01-01

A highly regiospecific synthesis of a series of indenoindoles is reported, together with X-ray studies and their activity against human prostate cancer cells PC-3 and LNCaP in vitro. The most effective compound 7,7-dimethyl-5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9bhexahydro-indeno[1,2-b]indole-9,10-dione 7q reduced the viability in both cell lines in a time and dose-dependent manner. Inhibitory effects were also observed on the adhesion, migration, and invasion of the prostate cancer cells as well as on clonogenic possibly by inhibition of MMP-9 activity. Molecular docking of 7q and 6k into MMP-9 human active site was also performed to determine the probable binding mode. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Novel dental adhesives containing nanoparticles of silver and amorphous calcium phosphate

PubMed Central

Melo, Mary Anne S.; Cheng, Lei; Zhang, Ke; Weir, Michael D.; Rodrigues, Lidiany K. A.; Xu, Hockin H. K.

2012-01-01

Objectives Secondary caries is the main reason for restoration failure, and replacement of the failed restorations accounts for 50–70% of all restorations. Antibacterial adhesives could inhibit residual bacteria in tooth cavity and invading bacteria along the margins. Calcium (Ca) and phosphate (P) ion release could remineralize the lesions. The objectives of this study were to incorporate nanoparticles of silver (NAg) and nanoparticles of amorphous calcium phosphate (NACP) into adhesive for the first time, and to investigate the effects on dentin bond strength and plaque microcosm biofilms. Methods Scotchbond Multi-Purpose adhesive was used as control. NAg were added into primer and adhesive at 0.1% by mass. NACP were mixed into adhesive at 10%, 20%, 30% and 40%. Microcosm biofilms were grown on disks with primer covering the adhesive on a composite. Biofilm metabolic activity, colony-forming units (CFU) and lactic acid were measured. Results Human dentin shear bond strengths (n=10) ranged from 26 to 34 MPa; adding NAg and NACP into adhesive did not decrease the bond strength (p > 0.1). SEM examination revealed resin tags from well-filled dentinal tubules. Numerous NACP infiltrated into the dentinal tubules. While NACP had little antibacterial effect, NAg in bonding agents greatly reduced the biofilm viability and metabolic activity, compared to the control (p < 0.05). CFU for total microorganisms, total streptococci, and mutans streptococci on bonding agents with NAg were an order of magnitude less than those of the control. Lactic acid production by biofilms for groups containing NAg was 1/4 of that of the control. Significance Dental plaque microcosm biofilm viability and acid production were greatly reduced on bonding agents containing NAg and NACP, without compromising dentin bond strength. The novel method of incorporating dual agents (remineralizing agent NACP and antibacterial agent NAg) may have wide applicability to other dental bonding systems. PMID:23138046

Blockage of hemichannels alters gene expression in osteocytes in a high magneto-gravitational environment

PubMed Central

Xu, Huiyun; Ning, Dandan; Zhao, Dezhi; Chen, Yunhe; Zhao, Dongdong; Gu, Sumin; Jiang, Jean X.; Shang, Peng

2017-01-01

Osteocytes, the most abundant cells in bone, are highly responsive to external environmental changes. We tested how Cx43 hemichannels which mediate the exchange of small molecules between cells and extracellular environment impact genome wide gene expression under conditions of abnormal gravity and magnetic field. To this end, we subjected osteocytic MLO-Y4 cells to a high magneto-gravitational environment and used microarray to examine global gene expression and a specific blocking antibody was used to assess the role of Cx43 hemichannels. While 3 hr exposure to abnormal gravity and magnetic field had relatively minor effects on global gene expression, blocking hemichannels significantly impacted the expression of a number of genes which are involved in cell viability, apoptosis, mineral absorption, protein absorption and digestion, and focal adhesion. Also, blocking of hemichannels enriched genes in multiple signaling pathways which are enaged by TGF-beta, Jak-STAT and VEGF. These results show the role of connexin hemichannels in bone cells in high magneto-gravitational environments. PMID:27814646

Laser surface treatment of polyamide and NiTi alloy and the effects on mesenchymal stem cell response

NASA Astrophysics Data System (ADS)

Waugh, D. G.; Lawrence, J.; Shukla, P.; Chan, C.; Hussain, I.; Man, H. C.; Smith, G. C.

2015-07-01

Mesenchymal stem cells (MSCs) are known to play important roles in development, post-natal growth, repair, and regeneration of mesenchymal tissues. What is more, surface treatments are widely reported to affect the biomimetic nature of materials. This paper will detail, discuss and compare laser surface treatment of polyamide (Polyamide 6,6), using a 60 W CO2 laser, and NiTi alloy, using a 100 W fiber laser, and the effects of these treatments on mesenchymal stem cell response. The surface morphology and composition of the polyamide and NiTi alloy were studied by scanning electron microscopy (SEM) and X-ray photoemission spectroscopy (XPS), respectively. MSC cell morphology cell counting and viability measurements were done by employing a haemocytometer and MTT colorimetric assay. The success of enhanced adhesion and spreading of the MSCs on each of the laser surface treated samples, when compared to as-received samples, is evidenced in this work.

Mapping cell surface adhesion by rotation tracking and adhesion footprinting

NASA Astrophysics Data System (ADS)

Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

2017-03-01

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

Compared to the amniotic membrane, Wharton's jelly may be a more suitable source of mesenchymal stem cells for cardiovascular tissue engineering and clinical regeneration.

PubMed

Pu, Lei; Meng, Mingyao; Wu, Jian; Zhang, Jing; Hou, Zongliu; Gao, Hui; Xu, Hui; Liu, Boyu; Tang, Weiwei; Jiang, Lihong; Li, Yaxiong

2017-03-21

The success of developing cardiovascular tissue engineering (CTE) grafts greatly needs a readily available cell substitute for endothelial and interstitial cells. Perinatal annexes have been proposed as a valuable source of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine. The objective of the present study is to evaluate the potential of human Wharton's jelly MSCs (WJ-MSCs) and amniotic membrane MSCs (AM-MSCs) as a seeding cell in CTE and cardiovascular regenerative medicine. WJ-MSCs/AM-MSCs were isolated and characterized in vitro according to their morphology, proliferation, self-renewal, phenotype, and multipotency. More importantly, the characteristics of hemocompatibility, extracellular matrix deposition, and gene expression and viability of both MSCs were investigated. Fibroblast-like human WJ-MSCs and AM-MSCs were successfully isolated and positively expressed the characteristic markers CD73, CD90, and CD105 but were negative for CD34, CD45, and HLA-DR. Both MSCs shared trilineage differentiation toward the adipogenic, osteogenic, and chondrogenic lineages. The proliferative and self-renewal capacity of WJ-MSCs was significantly higher than that of AM-MSCs (P < 0.001). WJ-MSCs provided comparable properties of antiplatelet adhesion and did not activate the coagulation cascade to endothelial cells. However, aggregated platelets were visualized on the surface of AM-MSCs-derived cell sheets and the intrinsic pathway was activated. Furthermore, WJ-MSCs have superior properties of collagen deposition and higher viability than AM-MSCs during cell sheet formation. This study highlights that WJ-MSCs could act as a functional substitute of endothelial and interstitial cells, which could serve as an appealing and practical single-cell source for CTE and regenerative therapy.

Cellular uptake of poly(allylamine hydrochloride) microcapsules with different deformability and its influence on cell functions.

PubMed

Yu, Wei; Zhang, Wenbo; Chen, Ying; Song, Xiaoxue; Tong, Weijun; Mao, Zhengwei; Gao, Changyou

2016-03-01

It is important to understand the safety issue and cell interaction pattern of polyelectrolyte microcapsules with different deformability before their use in biomedical applications. In this study, SiO2, poly(sodium-p-styrenesulfonate) (PSS) doped CaCO3 and porous CaCO3 spheres, all about 4μm in diameter, were used as templates to prepare microcapsules with different inner structure and subsequent deformability. As a result, three kinds of covalently assembled poly(allylaminehydrochloride)/glutaraldehyde (PAH/GA) microcapsules with similar size but different deformability under external osmotic pressure were prepared. The impact of different microcapsules on cell viability and functions are studied using smooth muscle cells (SMCs), endothelial cells (ECs) and HepG2 cells. The results demonstrated that viabilities of SMCs, ECs and HepG2 cells were not significantly influenced by either of the three kinds of microcapsules. However, the adhesion ability of SMCs and ECs as well as the mobility of SMCs, ECs and HepG2 cells were significantly impaired after treatment with microcapsules in a deformability dependent manner, especially the microcapsules with lower deformability caused higher impairment on cell functions. The cellular uptake kinetics, uptake pathways, intracellular distribution of microcapsules are further investigated in SMCs to reveal the potential mechanism. The SMCs showed faster uptake rate and exocytosis rate of microcapsules with lower deformability (Cap@CaCO3/PSS and Cap@CaCO3), leading to higher intracellular accumulation of microcapsules with lower deformability and possibly larger retardation of cell functions. The results pointed out that the deformability of microcapsules is an important factor governing the biological performance of microcapsules, which requires careful adjustment for further biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Up-regulation of tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 in human colon cancer Caco-2 cells following repetitive exposure to dietary levels of a polyphenol-rich chokeberry juice.

PubMed

Bermúdez-Soto, María J; Larrosa, Mar; Garcia-Cantalejo, Jesús M; Espín, Juan C; Tomás-Barberan, Francisco A; García-Conesa, María T

2007-04-01

Consumption of berries and red fruits rich in polyphenols may contribute to the reduction of colon cancer through mechanisms not yet understood. In this study, we investigated the response of subconfluent Caco-2 cells (a human colon carcinoma model) to repetitive exposure (2 h a day for a 4-day period) of a subtoxic dose of a chokeberry (Aronia melanocarpa) juice containing mixed polyphenols. To mimic physiological conditions, we subjected the chokeberry juice to in vitro gastric and pancreatic digestion. The effects on viability, proliferation and cell cycle were determined, and changes in the expression of genes in response to the chokeberry treatment were screened using Affymetrix oligonucleotide microarrays. Exposure to the chokeberry juice inhibited Caco-2 cell proliferation by causing G(2)/M cell cycle arrest. We detected changes in the expression of a group of genes involved in cell growth and proliferation and cell cycle regulation, as well as those associated to colorectal cancer. A selection of these genes was further confirmed by quantitative RT-PCR. Among these, the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), whose expression is known to be reduced in the majority of early adenomas and carcinomas, was up-regulated by the treatment both at the mRNA and protein levels (as shown by flow cytometry analysis). CEACAM1, with a significant regulatory role on cell proliferation of particular interest at early stages of cancer development, may be a potential target for chemoprevention by food components such as those present in polyphenol-rich fruits.

Cell proliferation on PVA/sodium alginate and PVA/poly(γ-glutamic acid) electrospun fiber.

PubMed

Yang, Jen Ming; Yang, Jhe Hao; Tsou, Shu Chun; Ding, Chian Hua; Hsu, Chih Chin; Yang, Kai Chiang; Yang, Chun Chen; Chen, Ko Shao; Chen, Szi Wen; Wang, Jong Shyan

2016-09-01

To overcome the obstacles of easy dissolution of PVA nanofibers without crosslinking treatment and the poor electrospinnability of the PVA cross-linked nanofibers via electrospinning process, the PVA based electrospun hydrogel nanofibers are prepared with post-crosslinking method. To expect the electrospun hydrogel fibers might be a promising scaffold for cell culture and tissue engineering applications, the evaluation of cell proliferation on the post-crosslinking electrospun fibers is conducted in this study. At beginning, poly(vinyl alcohol) (PVA), PVA/sodium alginate (PVASA) and PVA/poly(γ-glutamic acid) (PVAPGA) electrospun fibers were prepared by electrospinning method. The electrospun PVA, PVASA and PVAPGA nanofibers were treated with post-cross-linking method with glutaraldehyde (Glu) as crosslinking agent. These electrospun fibers were characterized with thermogravimetry analysis (TGA) and their morphologies were observed with a scanning electron microscope (SEM). To support the evaluation and explanation of cell growth on the fiber, the study of 3T3 mouse fibroblast cell growth on the surface of pure PVA, SA, and PGA thin films is conducted. The proliferation of 3T3 on the electrospun fiber surface of PVA, PVASA, and PVAPGA was evaluated by seeding 3T3 fibroblast cells on these crosslinked electrospun fibers. The cell viability on electrospun fibers was conducted with water-soluble tetrazolium salt-1 assay (Cell Proliferation Reagent WST-1). The morphology of the cells on the fibers was also observed with SEM. The results of WST-1 assay revealed that 3T3 cells cultured on different electrospun fibers had similar viability, and the cell viability increased with time for all electrospun fibers. From the morphology of the cells on electrospun fibers, it is found that 3T3 cells attached on all electrospun fiber after 1day seeded. Cell-cell communication was noticed on day 3 for all electrospun fibers. Extracellular matrix (ECM) productions were found and cell-ECM adhesion was shown on day 7. The cell number was also increased on all of the crosslinked electrospun fibers. It seems that the PVA based electrospun hydrogel nanofibers prepared with post-crosslinking method can be used as scaffold for tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

Phenolic Acid Composition, Antiatherogenic and Anticancer Potential of Honeys Derived from Various Regions in Greece

PubMed Central

Spilioti, Eliana; Jaakkola, Mari; Tolonen, Tiina; Lipponen, Maija; Virtanen, Vesa; Chinou, Ioanna; Kassi, Eva; Karabournioti, Sofia; Moutsatsou, Paraskevi

2014-01-01

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content. PMID:24752205

Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

PubMed

Soriano-Souza, Carlos Alberto; Rossi, Andre L; Mavropoulos, Elena; Hausen, Moema A; Tanaka, Marcelo N; Calasans-Maia, Mônica D; Granjeiro, Jose M; Rocha-Leão, Maria Helena M; Rossi, Alexandre M

2015-04-01

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 μgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

PubMed Central

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

Evaluation of the effect of supplementing fermented milk with quinoa flour on probiotic activity.

PubMed

Casarotti, Sabrina N; Carneiro, Bruno M; Penna, Ana Lúcia B

2014-10-01

In this work, we investigated the effect of supplementing fermented milk with quinoa flour as an option to increase probiotic activity during fermented milk production and storage. Fermented milk products were produced with increasing concentrations of quinoa flour (0, 1, 2, or 3g/100g) and submitted to the following analyses at 1, 14, and 28 d of refrigerated storage: postacidification, bacterial viability, resistance of probiotics to simulated gastrointestinal (GI) conditions, and adhesion of probiotics to Caco-2 cells in vitro. The kinetics of acidification were measured during the fermentation process. The time to reach maximum acidification rate, time to reach pH 5.0, and time to reach pH 4.6 (end of fermentation) were similar for all treatments. Adding quinoa flour had no effect on fermentation time; however, it did contribute to postacidification of the fermented milk during storage. Quinoa flour did not affect counts of Bifidobacterium animalis ssp. lactis BB-12 or Lactobacillus acidophilus La-5 during storage, it did not protect the probiotic strains during simulated GI transit, and it did not have a positive effect on the adhesion of probiotic bacteria to Caco-2 cells in vitro. Additionally, the adhesion of strains to Caco-2 cells decreased during refrigerated storage of fermented milk. Although the addition of up to 3% quinoa flour had a neutral effect on probiotic activity, its incorporation to fermented milk can be recommended because it is an ingredient with high nutritive value, which may increase the appeal of the product to consumers. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A Review of Cell Adhesion Studies for Biomedical and Biological Applications.

PubMed

Khalili, Amelia Ahmad; Ahmad, Mohd Ridzuan

2015-08-05

Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artificial bone and tooth replacement, the development of tissue-on-a-chip and organ-on-a-chip in tissue engineering, the effects of biochemical treatments and environmental stimuli to the cell adhesion, the potential of drug treatments, cancer metastasis study, and the determination of the adhesion properties of normal and cancerous cells. This review discussed the overview of the available methods to study cell adhesion through attachment and detachment events.

A Review of Cell Adhesion Studies for Biomedical and Biological Applications

PubMed Central

Ahmad Khalili, Amelia; Ahmad, Mohd Ridzuan

2015-01-01

Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artificial bone and tooth replacement, the development of tissue-on-a-chip and organ-on-a-chip in tissue engineering, the effects of biochemical treatments and environmental stimuli to the cell adhesion, the potential of drug treatments, cancer metastasis study, and the determination of the adhesion properties of normal and cancerous cells. This review discussed the overview of the available methods to study cell adhesion through attachment and detachment events. PMID:26251901

Titanium dioxide nanotubes addition to self-adhesive resin cement: Effect on physical and biological properties.

PubMed

Ramos-Tonello, Carla M; Lisboa-Filho, Paulo N; Arruda, Larisa B; Tokuhara, Cintia K; Oliveira, Rodrigo C; Furuse, Adilson Y; Rubo, José H; Borges, Ana Flávia S

2017-07-01

This study has investigated the influence of Titanium dioxide nanotubes (TiO 2 -nt) addition to self-adhesive resin cement on the degree of conversion, water sorption, and water solubility, mechanical and biological properties. A commercially available auto-adhesive resin cement (RelyX U200™, 3M ESPE) was reinforced with varying amounts of nanotubes (0.3, 0.6, 0.9wt%) and evaluated at different curing modes (self- and dual cure). The DC in different times (3, 6, 9, 12 and 15min), water sorption (Ws) and solubility (Sl), 3-point flexural strength (σf), elastic modulus (E), Knoop microhardness (H) and viability of NIH/3T3 fibroblasts were performed to characterize the resin cement. Reinforced self-adhesive resin cement, regardless of concentration, increased the DC for the self- and dual-curing modes at all times studied. The concentration of the TiO 2 -nt and the curing mode did not influence the Ws and Sl. Regarding σf, concentrations of both 0.3 and 0.9wt% for self-curing mode resulted in data similar to that of dual-curing unreinforced cement. The E increased with the addition of 0.9wt% for self-cure mode and H increased with 0.6 and 0.9wt% for both curing modes. Cytotoxicity assays revealed that reinforced cements were biocompatible. TiO 2 -nt reinforced self-adhesive resin cement are promising materials for use in indirect dental restorations. Taken together, self-adhesive resin cement reinforced with TiO 2 -nt exhibited physicochemical and mechanical properties superior to those of unreinforced cements, without compromising their cellular viability. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

In vitro Flow Adhesion Assay for Analyzing Shear-resistant Adhesion of Metastatic Cancer Cells to Endothelial Cells.

PubMed

Kang, Shin-Ae; Bajana, Sandra; Tanaka, Takemi

2016-02-20

Hematogenous metastasis is a primary cause of mortality from metastatic cancer. The shear-resistant adhesion of circulating tumor cells to the vascular endothelial cell surface under blood flow is an essential step in cell extravasation and further tissue invasion. This is similar to a process exploited by leukocytes for adhesion to inflamed blood vessels (leukocyte mimicry). The shear resistant adhesion is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligand expressed on circulating cells. Thus, weak interaction results in a rapid detachment of circulating cells from endothelium. Despite the critical role of vascular adhesion of cancer cells in hematogenous metastasis, our knowledge regarding this process has been limited due to the difficulty of mimicking dynamic flow conditions in vitro . In order to gain better insight into the shear-resistant adhesion of cancer cells to the endothelium, we developed a protocol for measuring the shear resistant adhesion of circulating tumor cells to endothelial cells under physiologic flow conditions by adapting a well established flow adhesion assay for inflammatory cells. This technique is useful to evaluate 1) the shear resistant adhesion competency of cancer cells and 2) the endothelial adhesion molecules necessary to support cancer cell adhesion (Kang et al. , 2015).

A novel and simple cell-based electrochemical impedance biosensor for evaluating the combined toxicity of DON and ZEN.

PubMed

Gu, Wenshu; Zhu, Pei; Jiang, Donglei; He, Xingxing; Li, Yun; Ji, Jian; Zhang, Lijuan; Sun, Yange; Sun, Xiulan

2015-08-15

In this study, a novel and simple cell-based electrochemical biosensor was developed to assess the individual and combined toxicity of deoxynivalenol (DON) and zearalenone (ZEN) on BEL-7402 cells. The sensor was fabricated by modification with AuNPs, p-aminothiophenol, and folic acid in succession. The BEL-7402 cells which had a good activity were adhered on the electrode through the high affinity between the folate receptor and folic acid selectivity. We used the collagen to maintain the cell adhesion and viability. Electrochemical impedance spectroscopy (EIS) was developed to evaluate the individual and combined toxicity of DON and ZEN. Our results indicate that DON and ZEN caused a marked decrease in the cell viability in a dose-dependent manner. The value of electrochemical impedance spectroscopy decreased with the concentration of DON and ZEN in range of 0.1-20, 0.1-50 μg/ml with the detection limit as 0.03, 0.05 μg/ml, respectively, the IC50 for DON and ZEN as obtained by the proposed electrochemical method were 7.1 μg/ml and 24.6 μg/ml, respectively, and the combination of two mycotoxins appears to generate an additive response. The electrochemical cytotoxicity evaluation result was confirmed by biological assays. Compared to conventional methods, this electrochemical test is inexpensive, highly sensitive, and fast to respond, with long-term monitoring and real-time measurements. The proposed method provides a new avenue for evaluating the toxicity of mycotoxins. Copyright © 2015 Elsevier B.V. All rights reserved.

Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

PubMed

Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

2015-03-01

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Successful vitrification of human amnion-derived mesenchymal stem cells.

PubMed

Moon, Jeong Hee; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun; Lim, Hyun Jung; Kim, Hae Kwon

2008-08-01

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering

PubMed Central

Parizek, Martin; Douglas, Timothy EL; Novotna, Katarina; Kromka, Alexander; Brady, Mariea A; Renzing, Andrea; Voss, Eske; Jarosova, Marketa; Palatinus, Lukas; Tesarek, Pavel; Ryparova, Pavla; Lisa, Věra; dos Santos, Ana M; Bacakova, Lucie

2012-01-01

Background Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering. Methods In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA). Results In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 μm2 versus 1.28 ± 0.09 μm2 in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1–7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity. Conclusion This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering. PMID:22619532

Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering.

PubMed

Parizek, Martin; Douglas, Timothy E L; Novotna, Katarina; Kromka, Alexander; Brady, Mariea A; Renzing, Andrea; Voss, Eske; Jarosova, Marketa; Palatinus, Lukas; Tesarek, Pavel; Ryparova, Pavla; Lisa, Věra; dos Santos, Ana M; Warnke, Patrick H; Bacakova, Lucie

2012-01-01

Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering. In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA). In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 μm(2) versus 1.28 ± 0.09 μm(2) in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1-7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity. This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering.

Polysialic acid immobilized on silanized glass surfaces: a test case for its use as a biomaterial for nerve regeneration.

PubMed

Steinhaus, Stephanie; Stark, Yvonne; Bruns, Stephanie; Haile, Yohannes; Scheper, Thomas; Grothe, Claudia; Behrens, Peter

2010-04-01

The immobilization of polysialic acid (polySia) on glass substrates has been investigated with regard to the applicability of this polysaccharide as a novel, biocompatible and bioresorbable material for tissue engineering, especially with regard to its use in nerve regeneration. PolySia, a homopolymer of alpha-2,8-linked sialic acid, is involved in post-translational modification of the neural cell adhesion molecule (NCAM). The degradation of polySia can be controlled which makes it an interesting material for coating and for scaffold construction in tissue engineering. Here, we describe the immobilization of polySia on glass surfaces via an epoxysilane linker. Whereas glass surfaces will not actually be used in nerve regeneration scaffolds, they provide a simple and efficient means for testing various methods for the investigation of immobilized polySia. The modified surfaces were investigated with contact angle measurements and the quantity of immobilized polySia was examined by the thiobarbituric acid assay and a specific polySia-ELISA. The interactions between the polySia-modified surface and immortalized Schwann cells were evaluated via cell adhesion and cell viability assays. The results show that polySia can be immobilized on glass surfaces via the epoxysilane linker and that surface-bound polySia has no toxic effects on Schwann cells. Therefore, as a key substance in the development of vertebrates and as a favourable substrate for the cultivation of Schwann cells, it offers interesting features for the use in nerve guidance tubes for treatment of peripheral nerve injuries.

Effects of the Food Manufacturing Chain on the Viability and Functionality of Bifidobacterium animalis through Simulated Gastrointestinal Conditions

PubMed Central

Jantama, Sirima Suvarnakuta; Prasitpuriprecha, Chutinun; Kanchanatawee, Sunthorn

2016-01-01

The viability and functionality of probiotics may be influenced by industrial production processes resulting in a decrease in probiotic efficiency that benefit the health of humans. This study aimed to investigate the probiotic characteristics of Bifidobacterium strains isolated from fecal samples of healthy Thai infants. In the present work, three local strains (BF014, BF052, and BH053) belonging to Bifidobacterium animalis showed a great resistance against conditions simulating the gastrointestinal tract. Among these, B. animalis BF052 possessed considerable probiotic properties, including high acid and bile tolerance, strong adhesion capability to Caco-2 cells, and inhibitory activity against pathogens including Salmonella typhimurium and Vibrio cholerae. This strain also exhibited a high survival rate compared to commercial strains during storage in a wide variety of products, including pasteurized milk, soy milk, drinking yogurt, and orange juice. The impact of food processing processes as well as the freeze-drying process, storage of freeze-dried powders, and incorporation of freeze-dried cells in food matrix on probiotic properties was also determined. The stability of the probiotic properties of the BF052 strain was not affected by food processing chain, especially its resistance in the simulated gastrointestinal conditions and its adherence ability to Caco-2 cells. It indicates that it satisfies the criteria as a potential probiotic and may be used as an effective probiotic starter in food applications. PMID:27333286

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

PubMed Central

Mick, Enrico

2014-01-01

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

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

PubMed

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

2014-01-01

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

Soluble adhesion molecules in human cancers: sources and fates.

PubMed

van Kilsdonk, Jeroen W J; van Kempen, Léon C L T; van Muijen, Goos N P; Ruiter, Dirk J; Swart, Guido W M

2010-06-01

Adhesion molecules endow tumor cells with the necessary cell-cell contacts and cell-matrix interactions. As such, adhesion molecules are involved in cell signalling, proliferation and tumor growth. Rearrangements in the adhesion repertoire allow tumor cells to migrate, invade and form metastases. Besides these membrane-bound adhesion molecules several soluble adhesion molecules are detected in the supernatant of tumor cell lines and patient body fluids. Truncated soluble adhesion molecules can be generated by several conventional mechanisms, including alternative splicing of mRNA transcripts, chromosomal translocation, and extracellular proteolytic ectodomain shedding. Secretion of vesicles (ectosomes and exosomes) is an alternative mechanism mediating the release of full-length adhesion molecules. Soluble adhesion molecules function as modulators of cell adhesion, induce proteolytic activity and facilitate cell signalling. Additionally, adhesion molecules present on secreted vesicles might be involved in the vesicle-target cell interaction. Based on currently available data, released soluble adhesion molecules contribute to cancer progression and therefore should not be regarded as unrelated and non-functional side products of tumor progression. 2010 Elsevier GmbH. All rights reserved.

The roles of cell adhesion molecules in tumor suppression and cell migration: a new paradox.

PubMed

Moh, Mei Chung; Shen, Shali

2009-01-01

In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.

Characterization of Selected Lactobacillus Strains for Use as Probiotics

PubMed Central

Song, Minyu; Yun, Bohyun; Moon, Jae-Hak; Park, Dong-June; Lim, Kwangsei; Oh, Sejong

2015-01-01

The aim of this study was to evaluate the functional properties of lactic acid bacteria from various sources and to identify strains for use as probiotics. Ten Lactobacillus strains were selected and their properties such as bile tolerance, acid resistance, cholesterol assimilation activity, and adherence to HT-29 cells were assessed to determine their potential as probiotics. Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, L. sakei CH8, and L. acidophilus M23 were found to show full tolerance to the 0.3% bile acid. All strains without L. acidophilus M23 were the most acid-tolerant strains. After incubating the strains at pH 2.5 for 2 h, their viability decreased by 3 Log cells. Some strains survived at pH 2.5 in the presence of pepsin and 0.3% bile acid. Lactobacillus sp. JNU 8829, L. acidophilus KU41, L. acidophilus M23, L. fermentum NS2, L. plantarum M13, and L. plantarum NS3 were found to reduce cholesterol levels by >50% in vitro. In the adhesion assay, Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, and L. sakei CH8 showed higher adhesion activities after 2 h of co-incubation with the intestinal cells. The results of this comprehensive analysis shows that this new probiotic strain named, Lactobacillus sp. JNU 8829 could be a promising candidate for dairy products. PMID:26761878

MARCKS promotes invasion and is associated with biochemical recurrence in prostate cancer

PubMed Central

Dorris, Emma; O'Neill, Amanda; Hanrahan, Karen; Treacy, Ann; Watson, R. William

2017-01-01

Background Overtreatment of low-grade prostate cancer is a recognised problem for clinicians and patients. However, under-treatment runs the risk of missing the opportunity for cure in those who could benefit. Identification of new biomarkers of disease progression, including metastases, is required to better stratify and appropriately treat these patients. The ability to predict if prostate cancer will recur is an important clinical question that would impact treatment options for patients. Studies in other cancers have associated MARCKS with metastasis. Methods Tissue microarrays of local prostatectomy samples from a cohort of biochemical recurrent and non-biochemical recurrent tumours were assayed for MARCKS protein expression. Prostate cancer cell lines were transfected with siRNA targeting MARCKS or a control and functional endpoints of migration, invasion, proliferation, viability and apoptosis were measured. Actin was visualised by fluorescent microscopy and evidence of a cadherin switch and activation of the AKT pathway were assayed. Results MARCKS was upregulated in biochemical recurrent patients compared to non-biochemical recurrent. Knockdown of MARCKS reduced migration and invasion of prostate cancer cells, reduced MMP9 mRNA expression, as well as decreasing cell spreading and increased cell:cell adhesion in prostate cancer cell colonies. Knockdown of MARCKS had no effect on proliferation, viability or apoptosis of the prostate cancer cells. Conclusions In conclusion, MARCKS promotes migration and invasion and is associated with biochemical recurrence in localised prostate cancer tumours. The mechanisms by which this occurs have yet to be fully elucidated but lack of a cadherin switch indicates it is not via epithelial-to-mesenchymal transition. Actin rearrangement indicates that MARCKS promotes invasion through regulating the architecture of the cell. PMID:29069765

A chip assisted immunomagnetic separation system for the efficient capture and in situ identification of circulating tumor cells.

PubMed

Tang, Man; Wen, Cong-Ying; Wu, Ling-Ling; Hong, Shao-Li; Hu, Jiao; Xu, Chun-Miao; Pang, Dai-Wen; Zhang, Zhi-Ling

2016-04-07

The detection of circulating tumor cells (CTCs), a kind of "liquid biopsy", represents a potential alternative to noninvasive detection, characterization and monitoring of carcinoma. Many previous studies have shown that the number of CTCs has a significant relationship with the stage of cancer. However, CTC enrichment and detection remain notoriously difficult because they are extremely rare in the bloodstream. Herein, aided by a microfluidic device, an immunomagnetic separation system was applied to efficiently capture and in situ identify circulating tumor cells. Magnetic nanospheres (MNs) were modified with an anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody to fabricate immunomagnetic nanospheres (IMNs). IMNs were then loaded into the magnetic field controllable microfluidic chip to form uniform IMN patterns. The IMN patterns maintained good stability during the whole processes including enrichment, washing and identification. Apart from its simple manufacture process, the obtained microfluidic device was capable of capturing CTCs from the bloodstream with an efficiency higher than 94%. The captured cells could be directly visualized with an inverted fluorescence microscope in situ by immunocytochemistry (ICC) identification, which decreased cell loss effectively. Besides that, the CTCs could be recovered completely just by PBS washing after removal of the permanent magnets. It was observed that all the processes showed negligible influence on cell viability (viability up to 93%) and that the captured cells could be re-cultured for more than 5 passages after release without disassociating IMNs. In addition, the device was applied to clinical samples and almost all the samples from patients showed positive results, which suggests it could serve as a valuable tool for CTC enrichment and detection in the clinic.

Role of caspase-9 in the effector caspases and genome expressions, and growth of bovine skeletal myoblasts.

PubMed

Van Ba, Hoa; Hwang, Inho

2014-02-01

Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48 h. Results indicated that all three siRNAs could silence the caspase-9 mRNA expression significantly. Particularly, the mRNA expression level of caspase-9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase-9 led to the significant increases in the mRNA and protein expressions of effector caspase-3, whereas the reduction in mRNA and protein expressions of caspase-7. The microarray results showed that the suppression of caspase-9 resulted in significant upregulations of cell proliferation-, adhesion-, growth-, development- and division-regulating genes, whereas the reduction in the expressions of cell death program- and stress response-regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase-9 could play an important role in the control of cell growth, and knockdown of caspase-9 may have genuine potential in the treatment of skeletal muscle atrophy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Thermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency.

PubMed

Rennert, Knut; Nitschke, Mirko; Wallert, Maria; Keune, Natalie; Raasch, Martin; Lorkowski, Stefan; Mosig, Alexander S

2017-01-01

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte-derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte-derived macrophages. In summary, we observed similar functionality and viability of primary monocyte-derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

PubMed

Nam, Seo Hee; Kang, Minkyung; Ryu, Jihye; Kim, Hye-Jin; Kim, Doyeun; Kim, Dae Gyu; Kwon, Nam Hoon; Kim, Sunghoon; Lee, Jung Weon

2016-04-01

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

Cell adhesion heterogeneity reinforces tumour cell dissemination: novel insights from a mathematical model.

PubMed

Reher, David; Klink, Barbara; Deutsch, Andreas; Voss-Böhme, Anja

2017-08-11

Cancer cell invasion, dissemination, and metastasis have been linked to an epithelial-mesenchymal transition (EMT) of individual tumour cells. During EMT, adhesion molecules like E-cadherin are downregulated and the decrease of cell-cell adhesion allows tumour cells to dissociate from the primary tumour mass. This complex process depends on intracellular cues that are subject to genetic and epigenetic variability, as well as extrinsic cues from the local environment resulting in a spatial heterogeneity in the adhesive phenotype of individual tumour cells. Here, we use a novel mathematical model to study how adhesion heterogeneity, influenced by intrinsic and extrinsic factors, affects the dissemination of tumour cells from an epithelial cell population. The model is a multiscale cellular automaton that couples intracellular adhesion receptor regulation with cell-cell adhesion. Simulations of our mathematical model indicate profound effects of adhesion heterogeneity on tumour cell dissemination. In particular, we show that a large variation of intracellular adhesion receptor concentrations in a cell population reinforces cell dissemination, regardless of extrinsic cues mediated through the local cell density. However, additional control of adhesion receptor concentration through the local cell density, which can be assumed in healthy cells, weakens the effect. Furthermore, we provide evidence that adhesion heterogeneity can explain the remarkable differences in adhesion receptor concentrations of epithelial and mesenchymal phenotypes observed during EMT and might drive early dissemination of tumour cells. Our results suggest that adhesion heterogeneity may be a universal trigger to reinforce cell dissemination in epithelial cell populations. This effect can be at least partially compensated by a control of adhesion receptor regulation through neighbouring cells. Accordingly, our findings explain how both an increase in intra-tumour adhesion heterogeneity and the loss of control through the local environment can promote tumour cell dissemination. This article was reviewed by Hanspeter Herzel, Thomas Dandekar and Marek Kimmel.

In vitro comparison of new bisphosphonic acids and zoledronate effects on human gingival fibroblasts viability, inflammation and matrix turnover.

PubMed

De Colli, Marianna; Tortorella, Paolo; Marconi, Guya Diletta; Agamennone, Mariangela; Campestre, Cristina; Tauro, Marilena; Cataldi, Amelia; Zara, Susi

2016-11-01

Bisphosphonates (BPs) are drugs clinically used in resorptive diseases. It was already proved that some clinically relevant BPs can inhibit a class of enzymes called matrix metalloproteinases (MMPs), required during tissue remodelling. Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present study was to compare the effect of zoledronic acid (ZA), the most potent bisphosphonate available as therapy, with new sulfonamide containing BPs in an in vitro model of human gingival fibroblasts (HGFs). Western blot was used to measure procollagen I, β1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability; L-lactate-dehydrogenase (LDH) measurement was performed for toxicity evaluation and ELISA for prostaglandin E 2 (PGE 2 ) secretion assessment. When compared with ZA, the treatment with the newly synthesized compounds shows increasing viability, procollagen I expression and decreased expression of β1 integrin in HGFs. Higher levels of released LDH, PGE 2 and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthesized compounds-treated samples. These findings allowed to conclude that new tested BPs did not affect HGFs viability and adhesion, did not induce cellular toxicity, were not responsible for inflammatory event induction and could preserve the physiological matrix turnover. It could be hypothesized that the new molecules were better tolerated by soft tissues, resulting in lesser side effects.

Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daniels, Candelaria C.; Rovnak, Joel; Quackenbush, Sandra L.

2008-06-05

Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assaysmore » and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKC{alpha}, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.« less

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro.

PubMed

Hyysalo, Anu; Ristola, Mervi; Mäkinen, Meeri E-L; Häyrynen, Sergei; Nykter, Matti; Narkilahti, Susanna

2017-10-01

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

In vitro and in vivo biocompatibility study on laser 3D microstructurable polymers

NASA Astrophysics Data System (ADS)

Malinauskas, Mangirdas; Baltriukiene, Daiva; Kraniauskas, Antanas; Danilevicius, Paulius; Jarasiene, Rasa; Sirmenis, Raimondas; Zukauskas, Albertas; Balciunas, Evaldas; Purlys, Vytautas; Gadonas, Roaldas; Bukelskiene, Virginija; Sirvydis, Vytautas; Piskarskas, Algis

2012-09-01

Films and microstructured scaffolds have been fabricated using direct laser writing out of different polymers: hybrid organic-inorganic ORMOCORE b59, acrylate-based AKRE23, novel organic-inorganic Zr containing hybrid SZ2080, and biodegradable PEG-DA-258. Adult myogenic stem cells were grown on these surfaces in vitro. Their adhesion, growth, and viability test results suggest good potential applicability of the materials in biomedical practice. Pieces of these polymers were implanted in rat's paravertebral back tissue. Histological examination of the implants and surrounding tissue ex vivo after 3 weeks of implantation was conducted and results show the materials to be at least as biocompatible as surgical clips or sutures. The applied direct laser writing technique seems to offer good future prospects in a polymeric 3D scaffold design for artificial tissue engineering with autologous stem cells.

Bonding and fusion of meniscus fibrocartilage using a novel chondroitin sulfate bone marrow tissue adhesive.

PubMed

Simson, Jacob A; Strehin, Iossif A; Allen, Brian W; Elisseeff, Jennifer H

2013-08-01

The weak intrinsic meniscus healing response and technical challenges associated with meniscus repair contribute to a high rate of repair failures and meniscectomies. Given this limited healing response, the development of biologically active adjuncts to meniscal repair may hold the key to improving meniscal repair success rates. This study demonstrates the development of a bone marrow (BM) adhesive that binds, stabilizes, and stimulates fusion at the interface of meniscus tissues. Hydrogels containing several chondroitin sulfate (CS) adhesive levels (30, 50, and 70 mg/mL) and BM levels (30%, 50%, and 70%) were formed to investigate the effects of these components on hydrogel mechanics, bovine meniscal fibrochondrocyte viability, proliferation, matrix production, and migration ability in vitro. The BM content positively and significantly affected fibrochondrocyte viability, proliferation, and migration, while the CS content positively and significantly affected adhesive strength (ranged from 60±17 kPa to 335±88 kPa) and matrix production. Selected material formulations were translated to a subcutaneous model of meniscal fusion using adhered bovine meniscus explants implanted in athymic rats and evaluated over a 3-month time course. Fusion of adhered meniscus occurred in only the material containing the highest BM content. The technology can serve to mechanically stabilize the tissue repair interface and stimulate tissue regeneration across the injury site.

Cell-cell and cell-extracellular matrix adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure.

PubMed

Goodwin, Katharine; Lostchuck, Emily E; Cramb, Kaitlyn M L; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo; Tanentzapf, Guy

2017-05-15

Tissue morphogenesis relies on the coordinated action of actin networks, cell-cell adhesions, and cell-extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell-cell and cell-ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell-cell and cell-ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell-cell and cell-ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell-ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell-cell adhesion caused up-regulation of cell-ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell-cell and cell-ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis. © 2017 Goodwin, Lostchuck, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Actin-Based Adhesion Modules Mediate Cell Interactions with the Extracellular Matrix and Neighboring Cells.

PubMed

Bachir, Alexia I; Horwitz, Alan Rick; Nelson, W James; Bianchini, Julie M

2017-07-05

Cell adhesions link cells to the extracellular matrix (ECM) and to each other and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping, functional modules. These modules establish physical associations with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as to sense and translate the mechanical properties of the cellular environment into changes in cell organization and behavior. Here, we review the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions and how adhesion molecules mediate cross talk between cell-ECM and cell-cell adhesion sites. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Autophagy promotes degradation of internalized collagen and regulates distribution of focal adhesions to suppress cell adhesion

PubMed Central

Kawano, Shinichi; Esaki, Motohiro; Torisu, Kumiko; Matsuno, Yuichi; Kitazono, Takanari

2017-01-01

ABSTRACT Adhesion of cells to the extracellular matrix (ECM) via focal adhesions (FAs) is crucial for cell survival, migration, and differentiation. Although the regulation of FAs, including by integrins and the ECM, is important to cell behavior, how FAs are regulated is not well known. Autophagy is induced by both cell adhesion and cell detachment. Here, we showed that autophagosomes are located close to internalized collagen and paxillin, which is a well-known marker of FAs. Autophagy-deficient cells showed increased levels of internalized collagen compared with control cells. Moreover, paxillin exhibited a more peripheral distribution and the area of paxillin was increased, and adhesion-induced focal adhesion kinase signaling was impaired and adhesion was enhanced, in autophagy-deficient cells. These results suggest that autophagy suppressed cell adhesion by regulating internalized ECM and FAs. PMID:28970230

Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin

PubMed Central

Oh, Jaeho; Edwards, Erin E.; McClatchey, P. Mason; Thomas, Susan N.

2015-01-01

ABSTRACT Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell–cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner. PMID:26349809

Targeted delivery of probiotics to enhance gastrointestinal stability and intestinal colonisation.

PubMed

Dodoo, Cornelius C; Wang, Jie; Basit, Abdul W; Stapleton, Paul; Gaisford, Simon

2017-09-15

The aim of this work was to assess the viability of some commercial probiotics after exposure to gastric acid and the possibility of modifying these formulations for delivery into the distal parts of the intestines. Gastrointestinal tolerance testing was conducted for three commercial probiotics and an in-house freeze-dried Lactobacillus acidophilus strain. The contents of the commercial products and the in-house freeze-dried strain were then loaded into capsules for site-specific delivery into the colon using the Phloral ® coating technology; the viability upon release was then ascertained. An assessment of the potential of these products to adhere to intestinal cells was also conducted. The results showed that all the commercial products contained the minimum number of probiotic strains as indicated on their respective packages. When gastric acid tolerance tests were performed on these products, all the commercial probiotics and the prepared freeze-dried strain demonstrated over 10 6 CFU reductions within 5min. When these were encapsulated for site-specific delivery into the distal parts of the gut, viabilities of approximately 90% were obtained after these capsules had been initially deposited in gastric acid for 2h. An evaluation of the ability of the probiotic formulations to adhere to intestinal cells demonstrated adhesion in the range 64-76% for the products evaluated. The need to target the delivery of probiotics into the intestines has been demonstrated here as this offers a greater potential for colonisation of the intestines once the harshness of the stomach has been overcome. Copyright © 2017 Elsevier B.V. All rights reserved.

Remineralization Property of an Orthodontic Primer Containing a Bioactive Glass with Silver and Zinc

PubMed Central

Lee, Seung-Min; Kim, In-Ryoung; Park, Bong-Soo; Ko, Ching-Chang; Son, Woo-Sung; Kim, Yong-Il

2017-01-01

White spot lesions (WSLs) are irreversible damages in orthodontic treatment due to excessive etching or demineralization by microorganisms. In this study, we conducted a mechanical and cell viability test to examine the antibacterial properties of 0.2% and 1% bioactive glass (BAG) and silver-doped and zinc-doped BAGs in a primer and evaluated their clinical applicability to prevent WSLs. The microhardness statistically significantly increased in the adhesive-containing BAG, while the other samples showed no statistically significant difference compared with the control group. The shear bond strength of all samples increased compared with that of the control group. The cell viability of the control and sample groups was similar within 24 h, but decreased slightly over 48 h. All samples showed antibacterial properties. Regarding remineralization property, the group containing 0.2% of the samples showed remineralization properties compared with the control group, but was not statistically significant; further, the group containing 1% of the samples showed a significant difference compared with the control group. Among them, the orthodontic bonding primer containing 1% silver-doped BAG showed the highest remineralization property. The new orthodontic bonding primer used in this study showed an antimicrobial effect, chemical remineralization effect, and WSL prevention as well as clinically applicable properties, both physically and biologically. PMID:29088092

FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.

PubMed

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.

FACS-Based Isolation, Propagation and Characterization of Mouse Embryonic Cardiomyocytes Based on VCAM-1 Surface Marker Expression

PubMed Central

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K.; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes. PMID:24386094

Carnosic acid inhibits the proliferation and migration capacity of human colorectal cancer cells

PubMed Central

BARNI, M.V.; CARLINI, M.J.; CAFFERATA, E.G.; PURICELLI, L.; MORENO, S.

2012-01-01

Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. The objective of this study was to examine whether carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., would inhibit the cell viability of three CRC cell lines: Caco-2, HT29 and LoVo in a dose-dependent manner, with IC50 values in the range of 24–96 μM. CA induced cell death by apoptosis in Caco-2 line after 24 h of treatment and inhibited cell adhesion and migration, possibly by reducing the activity of secreted proteases such as urokinase plasminogen activator (uPA) and metalloproteinases (MMPs). These effects may be associated through a mechanism involving the inhibition of the COX-2 pathway, because we have determined that CA downregulates the expression of COX-2 in Caco-2 cells at both the mRNA and protein levels. Therefore, CA modulates different targets involved in the development of CRC. These findings indicate that carnosic acid may have anticancer activity and may be useful as a novel chemotherapeutic agent. PMID:22246562

Light responsive hybrid nanofibres for on-demand therapeutic drug and cell delivery.

PubMed

Li, Yan-Fang; Slemming-Adamsen, Peter; Wang, Jing; Song, Jie; Wang, Xueqin; Yu, Ying; Dong, Mingdong; Chen, Chunying; Besenbacher, Flemming; Chen, Menglin

2017-08-01

Smart materials for on-demand delivery of therapeutically active agents are challenging in pharmaceutical and biomaterials science. In the present study, we report hybrid nanofibres capable of being reversibly controlled to pulsatile deliver both therapeutic drugs and cells on-demand of near-infrared (NIR) light. The nanofibres, fabricated by co-electrospinning of poly (N-isopropylacrylamide), silica-coated gold nanorods and polyhedral oligomeric silsesquinoxanes have, for the first time, demonstrated rapid, reversible large-volume changes of 83% on-demand with NIR stimulation, with retained nanofibrous morphology. Combining with the extracellular matrix-mimicking fibrillary properties, the nanofibres achieved accelerated release of model drug or cells on demand with NIR triggering. The release of the model drug doxorubicin demonstrated normal anti-cancer efficacy by reducing the viability of human cervical cancer HeLa cells by 97% in 48 h. In parallel, the fibres allowed model cell NIH3T3 fibroblast entrapment, adhesion, proliferation, differentiation and, upon NIR irradiation, cell release with undisturbed cellular function. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Magnetostatic Field System for Uniform Cell Cultures Exposure

PubMed Central

Vergallo, Cristian; Piccoli, Claudia; Romano, Alberto; Panzarini, Elisa; Serra, Antonio; Manno, Daniela; Dini, Luciana

2013-01-01

The aim of the present work has been the design and the realization of a Magnetostatic Field System for Exposure of Cell cultures (MaFiSEC) for the uniform and the reproducible exposure of cell cultures to static magnetic fields (SMFs) of moderate magnetic induction. Experimental and computer-simulated physical measurements show that MaFiSEC: i) generates a SMF with magnetic induction that can be chosen in the range of 3 to 20 mT; ii) allows the uniform SMF exposure of cells growing in adhesion and in suspension; iii) is cheap and easy to use. The efficacy and reproducibility of MaFiSEC has been tested by comparing the biological effects exerted on isolated human lymphocytes by 72 h of exposure to a magnet (i.e. Neodymium Magnetic Disk, NMD) placed under the culture Petri dish. Lymphocytes morphology, viability, cell death, oxidative stress and lysosomes activity were the parameters chosen to evaluate the SMF biological effects. The continuous exposure of cells to a uniform SMF, achieved with MaFiSEC, allows highly reproducible biochemical and morphological data. PMID:23977284

Antibacterial abilities and biocompatibilities of Ti-Ag alloys with nanotubular coatings.

PubMed

Liu, Xingwang; Tian, Ang; You, Junhua; Zhang, Hangzhou; Wu, Lin; Bai, Xizhuang; Lei, Zeming; Shi, Xiaoguo; Xue, Xiangxin; Wang, Hanning

To endow implants with both short- and long-term antibacterial activities without impairing their biocompatibility, novel Ti-Ag alloy substrates with different proportions of Ag (1, 2, and 4 wt% Ag) were generated with nanotubular coverings (TiAg-NT). Unlike commercial pure Ti and titania nanotube, the TiAg-NT samples exhibited short-term antibacterial activity against Staphylococcus aureus ( S. aureus ), as confirmed by scanning electron microscopy and double staining with SYTO 9 and propidium iodide. A film applicator coating assay and a zone of inhibition assay were performed to investigate the long-term antibacterial activities of the samples. The cellular viability and cytotoxicity were evaluated through a Cell Counting Kit-8 assay. Annexin V-FITC/propidium iodide double staining was used to assess the level of MG63 cell apoptosis on each sample. All of the TiAg-NT samples, particularly the nanotube-coated Ti-Ag alloy with 2 wt% Ag (Ti2%Ag-NT), could effectively inhibit bacterial adhesion and kill the majority of adhered S. aureus on the first day of culture. Additionally, the excellent antibacterial abilities exhibited by the TiAg-NT samples were sustained for at least 30 days. Although Ti2%Ag-NT had less biocompatibility than titania nanotube, its performance was satisfactory, as demonstrated by the higher cellular viability and lower cell apoptosis rate obtained with it compared with those achieved with commercial pure Ti. The Ti1%Ag-NT and Ti4%Ag-NT samples did not yield good cell viability. This study indicates that the TiAg-NT samples can prevent biofilm formation and maintain their antibacterial ability for at least 1 month. Ti2%Ag-NT exhibited better antibacterial ability and biocompatibility than commercial pure Ti, which could be attributed to the synergistic effect of the presence of Ag (2 wt%) and the morphology of the nanotubes. Ti2%Ag-NT may offer a potential implant material that is capable of preventing implant-related infection.

Antibacterial abilities and biocompatibilities of Ti–Ag alloys with nanotubular coatings

PubMed Central

Liu, Xingwang; Tian, Ang; You, Junhua; Zhang, Hangzhou; Wu, Lin; Bai, Xizhuang; Lei, Zeming; Shi, Xiaoguo; Xue, Xiangxin; Wang, Hanning

2016-01-01

Purpose To endow implants with both short- and long-term antibacterial activities without impairing their biocompatibility, novel Ti–Ag alloy substrates with different proportions of Ag (1, 2, and 4 wt% Ag) were generated with nanotubular coverings (TiAg-NT). Methods Unlike commercial pure Ti and titania nanotube, the TiAg-NT samples exhibited short-term antibacterial activity against Staphylococcus aureus (S. aureus), as confirmed by scanning electron microscopy and double staining with SYTO 9 and propidium iodide. A film applicator coating assay and a zone of inhibition assay were performed to investigate the long-term antibacterial activities of the samples. The cellular viability and cytotoxicity were evaluated through a Cell Counting Kit-8 assay. Annexin V-FITC/propidium iodide double staining was used to assess the level of MG63 cell apoptosis on each sample. Results All of the TiAg-NT samples, particularly the nanotube-coated Ti–Ag alloy with 2 wt% Ag (Ti2%Ag-NT), could effectively inhibit bacterial adhesion and kill the majority of adhered S. aureus on the first day of culture. Additionally, the excellent antibacterial abilities exhibited by the TiAg-NT samples were sustained for at least 30 days. Although Ti2%Ag-NT had less biocompatibility than titania nanotube, its performance was satisfactory, as demonstrated by the higher cellular viability and lower cell apoptosis rate obtained with it compared with those achieved with commercial pure Ti. The Ti1%Ag-NT and Ti4%Ag-NT samples did not yield good cell viability. Conclusion This study indicates that the TiAg-NT samples can prevent biofilm formation and maintain their antibacterial ability for at least 1 month. Ti2%Ag-NT exhibited better antibacterial ability and biocompatibility than commercial pure Ti, which could be attributed to the synergistic effect of the presence of Ag (2 wt%) and the morphology of the nanotubes. Ti2%Ag-NT may offer a potential implant material that is capable of preventing implant-related infection. PMID:27843315

Effects of TiO2 and Co3O4 Nanoparticles on Circulating Angiogenic Cells

PubMed Central

Spigoni, Valentina; Cito, Monia; Alinovi, Rossella; Pinelli, Silvana; Passeri, Giovanni; Zavaroni, Ivana; Goldoni, Matteo; Campanini, Marco; Aliatis, Irene; Mutti, Antonio

2015-01-01

Background and Aim Sparse evidence suggests a possible link between exposure to airborne nanoparticles (NPs) and cardiovascular (CV) risk, perhaps through mechanisms involving oxidative stress and inflammation. We assessed the effects of TiO2 and Co3O4 NPs in human circulating angiogenic cells (CACs), which take part in vascular endothelium repair/replacement. Methods CACs were isolated from healthy donors’ buffy coats after culturing lymphomonocytes on fibronectin-coated dishes in endothelial medium for 7 days. CACs were pre-incubated with increasing concentration of TiO2 and Co3O4 (from 1 to 100 μg/ml) to test the effects of NP – characterized by Transmission Electron Microscopy – on CAC viability, apoptosis (caspase 3/7 activation), function (fibronectin adhesion assay), oxidative stress and inflammatory cytokine gene expression. Results Neither oxidative stress nor cell death were associated with exposure to TiO2 NP (except at the highest concentration tested), which, however, induced a higher pro-inflammatory effect compared to Co3O4 NPs (p<0.01). Exposure to Co3O4 NPs significantly reduced cell viability (p<0.01) and increased caspase activity (p<0.01), lipid peroxidation end-products (p<0.05) and pro-inflammatory cytokine gene expression (p<0.05 or lower). Notably, CAC functional activity was impaired after exposure to both TiO2 (p<0.05 or lower) and Co3O4 (p<0.01) NPs. Conclusions In vitro exposure to TiO2 and Co3O4 NPs exerts detrimental effects on CAC viability and function, possibly mediated by accelerated apoptosis, increased oxidant stress (Co3O4 NPs only) and enhancement of inflammatory pathways (both TiO2 and Co3O4 NPs). Such adverse effects may be relevant for a potential role of exposure to TiO2 and Co3O4 NPs in enhancing CV risk in humans. PMID:25803285

Biocorrosion behavior and cell viability of adhesive polymer coated magnesium based alloys for medical implants

NASA Astrophysics Data System (ADS)

Abdal-hay, Abdalla; Dewidar, Montasser; Lim, Jae Kyoo

2012-11-01

The present study was ultimately aimed to design novel adhesive biodegradable polymer, poly(vinyl acetate) (PVAc), coatings onto Mg based alloys by the dip-coating technique in order to control the degradation rate and enhance the biocompatibility of magnesium alloys. The influence of various solvents on PVAc surface topography and their protection of Mg alloys were dramatically studied in vitro. Electrochemical polarization, degradation, and PVAc film cytocompatibility were also tested. Our results showed that the solvent had a significant effect on coating quality. PVAc/dichloromethane solution showed a porous structure and solution concentration could control the porous size. The coatings prepared using tetrahydrofuran and dimethylformamide solvents are exceptional in their ability to generate porous morphology even at low polymer concentration. In general, the corrosion performance appears to be different on different PVAc-solvent system. Immersion tests illustrated that the porous morphology on PVAc stabilized corrosion rates. A uniform corrosion attack in artificial simulation body fluid was also exhibited. The cytocompatibility of osteoblast cells (MC3T3) revealed high adherence, proliferation, and survival on the porous structure of PVAc coated Mg alloy, which was not observed for the uncoated samples. This novel PVAc coating is a promising candidate for biodegradable implant materials, which might widen the use of Mg based implants.

Effect of Surface Modifications of Ti40Zr10Cu38Pd12 Bulk Metallic Glass and Ti-6Al-4V Alloy on Human Osteoblasts In Vitro Biocompatibility

PubMed Central

Blanquer, Andreu; Hynowska, Anna; Nogués, Carme; Ibáñez, Elena; Sort, Jordi; Baró, Maria Dolors; Özkale, Berna; Pané, Salvador; Pellicer, Eva

2016-01-01

The use of biocompatible materials, including bulk metallic glasses (BMGs), for tissue regeneration and transplantation is increasing. The good mechanical and corrosion properties of Ti40Zr10Cu38Pd12 BMG and its previously described biocompatibility makes it a potential candidate for medical applications. However, it is known that surface properties like topography might play an important role in regulating cell adhesion, proliferation and differentiation. Thus, in the present study, Ti40Zr10Cu38Pd12 BMG and Ti6-Al-4V alloy were surface-modified electrochemically (nanomesh) or physically (microscratched) to investigate the effect of material topography on human osteoblasts cells (Saos-2) adhesion, proliferation and differentiation. For comparative purposes, the effect of mirror-like polished surfaces was also studied. Electrochemical treatments led to a highly interconnected hierarchical porous structure rich in oxides, which have been described to improve corrosion resistance, whereas microscratched surfaces showed a groove pattern with parallel trenches. Cell viability was higher than 96% for the three topographies tested and for both alloy compositions. In all cases, cells were able to adhere, proliferate and differentiate on the alloys, hence indicating that surface topography plays a minor role on these processes, although a clear cell orientation was observed on microscratched surfaces. Overall, our results provide further evidence that Ti40Zr10Cu38Pd12 BMG is an excellent candidate, in the present two topographies, for bone repair purposes. PMID:27243628

Piperine treatment suppresses Helicobacter pylori toxin entry in to gastric epithelium and minimizes β-catenin mediated oncogenesis and IL-8 secretion in vitro

PubMed Central

Tharmalingam, Nagendran; Park, Min; Lee, Min Ho; Woo, Hyun Jun; Kim, Hyun Woo; Yang, Ji Yeong; Rhee, Ki-Jong; Kim, Jong-Bae

2016-01-01

Helicobacter pylori related gastric cancer initiation has been studied widely. The objective of our present study was to evaluate the effect of a single compound piperine on H. pylori infection and its anti-inflammatory and anti-cancer effects in vitro. Cytotoxicity was tested by Ez-cytox cell viability assay kit. Effects of piperine on H. pylori toxin gene expression and IL-8 expression in mammalian cells during infection were assessed by RT-PCR. Effects of piperine on toxin entry into host cells, E-cadherin cleavage by H. pylori, and the changes in H. pylori mediated β-catenin expression and IL-8 secretion were determined by immunoblotting. Piperine treatment restrained the entry of CagA and VacA into AGS cells. Piperine administration in H. pylori infection reduced E-cadherin cleavage in stomach epithelium. In addition, H. pylori induced β-catenin up-regulation was reduced. Piperine administration impaired IL-8 secretion in H. pylori-infected gastric epithelial cells. As we reported previously piperine restrained H. pylori motility. The possible reason behind the H. pylori inhibition mechanism of piperine could be the dwindled motility, which weakened H. pylori adhesion to gastric epithelial cells. The reduced adhesion decreased the toxin entry thereby secreting less amount of IL-8. In addition, piperine treatment suppressed H. pylori protease led to reduction of E-cadherin cleavage and β-catenin expression resulting in diminished β-catenin translocation into the nucleus thus decreasing the risk of oncogenesis. To our knowledge, this is the preliminary report of piperine mediated H. pylori infection control on gastric epithelial cells in-vitro. PMID:27158376

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

PubMed

Wang, Y; Baumrucker, C R

2010-07-01

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P<0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P<0.05). The MeBo cells exhibited some inhibition with these natural ligands (P<0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P<0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P<0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P<0.05) and inhibited BME-UV1 cell viability (P<0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P<0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

Changes in E-cadherin rigidity sensing regulate cell adhesion.

PubMed

Collins, Caitlin; Denisin, Aleksandra K; Pruitt, Beth L; Nelson, W James

2017-07-18

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells into Motor Neuron-Like Cells on Three-Dimensional Collagen-Grafted Nanofibers.

PubMed

Bagher, Zohreh; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Mirzadeh, Hamid; Solouk, Atefeh; Soleimani, Mansooreh; Ai, Jafar; Nourani, Mohammad Reza; Joghataei, Mohammad Taghi

2016-05-01

Cell transplantation strategies have provided potential therapeutic approaches for treatment of neurodegenerative diseases. Mesenchymal stem cells from Wharton's jelly (WJMSCs) are abundant and available adult stem cells with low immunological incompatibility, which could be considered for cell replacement therapy in the future. However, MSC transplantation without any induction or support material causes poor control of cell viability and differentiation. In this study, we investigated the effect of the nanoscaffolds on WJMSCs differentiation into motor neuronal lineages in the presence of retinoic acid (RA) and sonic hedgehog (Shh). Surface properties of scaffolds have been shown to significantly influence cell behaviors such as adhesion, proliferation, and differentiation. Therefore, polycaprolactone (PCL) nanofibers were constructed via electrospinning, surface modified by plasma treatment, and grafted by collagen. Characterization of the scaffolds by means of ATR-FTIR, contact angel, and Bradford proved grafting of the collagen on the surface of the scaffolds. WJMSCs were seeded on nanofibrous and tissue culture plate (TCP) and viability of WJMSCs were measured by MTT assay and then induced to differentiate into motor neuron-like cells for 15 days. Differentiated cells were evaluated morphologically, and real-time PCR and immunocytochemistry methods were done to evaluate expression of motor neuron-like cell markers in mRNA and protein levels. Our results showed that obtained cells could express motor neuron biomarkers at both RNA and protein levels, but the survival and differentiation of WJMSCs into motor neuron-like cells on the PCL/collagen scaffold were higher than cultured cells in the TCP and PCL groups. Taken together, WJMSCs are an attractive stem cell source for inducing into motor neurons in vitro especially when grown on nanostructural scaffolds and PCL/collagen scaffolds can provide a suitable, three-dimensional situation for neuronal survival and differentiation that suggest their potential application towards nerve regeneration.

Tobacco and e-cigarette products initiate Kupffer cell inflammatory responses.

PubMed

Rubenstein, David A; Hom, Sarah; Ghebrehiwet, Berhane; Yin, Wei

2015-10-01

Kupffer cells are liver resident macrophages that are responsible for screening and clearing blood of pathogens and foreign particles. It has recently been shown that Kupffer cells interact with platelets, through an adhesion based mechanism, to aid in pathogen clearance and then these platelets re-enter the general systemic circulation. Thus, a mechanism has been identified that relates liver inflammation to possible changes in the systemic circulation. However, the role that Kupffer cells play in cardiovascular disease initiation/progression has not been elucidated. Thus, our objective was to determine whether or not Kupffer cells are responsive to a classical cardiovascular risk factor and if these changes can be transmitted into the general systemic circulation. If Kupffer cells initiate inflammatory responses after exposure to classical cardiovascular risk factors, then this provides a potential alternative/synergistic pathway for cardiovascular disease initiation. We aimed to elucidate the prevalence of this potential pathway. We hypothesized that Kupffer cells would initiate a robust inflammatory response after exposure to tobacco cigarette or e-cigarette products and that the inflammatory response would have the potential to antagonize other salient cells for cardiovascular disease progression. To test this, Kupffer cells were incubated with tobacco smoke extracts, e-cigarette vapor extracts or pure nicotine. Complement deposition onto Kupffer cells, Kupffer cell complement receptor expression, oxidative stress production, cytokine release and viability and density were assessed after the exposure. We observed a robust inflammatory response, oxidative stress production and cytokine release after Kupffer cells were exposed to tobacco or e-cigarette extracts. We also observed a marginal decrease in cell viability coupled with a significant decrease in cell density. In general, this was not a function of the extract formulation (e.g. tobacco vs. e-cigarette products or the formulation of the cigarette product). These results indicate that Kupffer cells are responsive to classical cardiovascular risk factors and that an inflammatory response is initiated that may pass into the general systemic circulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

There are four dynamically and functionally distinct populations of E-cadherin in cell junctions

PubMed Central

Erami, Zahra; Timpson, Paul; Yao, Wu; Zaidel-Bar, Ronen; Anderson, Kurt I.

2015-01-01

ABSTRACT E-cadherin is a trans-membrane tumor suppressor responsible for epithelial cell adhesion. E-cadherin forms adhesive clusters through combined extra-cellular cis- and trans-interactions and intracellular interaction with the actin cytoskeleton. Here we identify four populations of E-cadherin within cell junctions based on the molecular interactions which determine their mobility and adhesive properties. Adhesive and non-adhesive populations of E-cadherin each consist of mobile and immobile fractions. Up to half of the E-cadherin immobilized in cell junctions is non-adhesive. Incorporation of E-cadherin into functional adhesions require all three adhesive interactions, with deletion of any one resulting in loss of effective cell-cell adhesion. Interestingly, the only interaction which could independently slow the diffusion of E-cadherin was the tail-mediated intra-cellular interaction. The adhesive and non-adhesive mobile fractions of E-cadherin can be distinguished by their sensitivity to chemical cross-linking with adhesive clusters. Our data define the size, mobility, and adhesive properties of four distinct populations of E-cadherin within cell junctions, and support association with the actin cytoskeleton as the first step in adhesion formation. PMID:26471767

Influence of zinc oxide quantum dots in the antibacterial activity and cytotoxicity of an experimental adhesive resin.

PubMed

Garcia, Isadora Martini; Leitune, Vicente Castelo Branco; Visioli, Fernanda; Samuel, Susana Maria Werner; Collares, Fabrício Mezzomo

2018-06-01

To evaluate the influence of zinc oxide quantum dots (ZnO QDs ) into an experimental adhesive resin regarding the antibacterial activity against Streptococcus mutans and the cytotoxicity against pulp fibroblasts. ZnO QDs were synthesized by sol-gel process and were incorporated into 2-hydroxyethyl methacrylate (HEMA). An experimental adhesive resin was formulated by mixing 66.6 wt.% bisphenol A glycol dimethacrylate (BisGMA) and 33.3 wt.% HEMA with a photoinitiator system as control group. HEMA containing ZnO QDs was used for test group formulation. For the antibacterial activity assay, a direct contact inhibition evaluation was performed with biofilm of Streptococcus mutans (NCTC 10449). The cytotoxicity assay was performed by Sulforhodamine B (SRB) colorimetric assay for cell density determination using pulp fibroblasts. Data were analyzed by Student's t-test (α = 0.05). The antibacterial activity assay indicated statistically significant difference between the groups (p = 0.003), with higher values of biofilm formation on the polymerized samples of control group and a reduction of more than 50% of biofilm formation on ZnO QDs group. No difference of pulp fibroblasts viability was found between the adhesives (p = 0.482). ZnO QDs provided antibacterial activity when doped into an experimental adhesive resin without cytotoxic effect for pulp fibroblasts. Thus, the use of ZnO QDs is a strategy to develop antibiofilm restorative polymers with non-agglomerated nanofillers. ZnO QDs are non-agglomerated nanoscale fillers for dental resins and may be a strategy to reduce biofilm formation at dentin/restoration interface with no cytotoxicity for pulp fibroblasts. Copyright © 2018 Elsevier Ltd. All rights reserved.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758).

PubMed

Borges, Alana A; Lira, Gabriela P O; Nascimento, Lucas E; Queiroz Neta, Luiza B; Santos, Maria V O; Oliveira, Moacir F; Silva, Alexandre R; Pereira, Alexsandra F

2018-04-01

Skin vitrification is a promising and alternative tool for the conservation of biodiversity, especially for wild mammals, such as collared peccaries. Several factors can affect the success of this procedure, such as the cryoprotectant solution used. Therefore, this study was carried out to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from the collared peccary, aiming to study the application in biobanking, where cellular use is not immediately required. Then, Dulbecco's modified Eagle's medium (DMEM) composed of 2.2 g/L sodium bicarbonate and 10% fetal bovine serum (FBS) was supplemented with 3.0 M ethylene glycol (EG) or 3.0 M dimethyl sulfoxide (DMSO) or 1.5 M EG plus 1.5 M DMSO with or without sucrose (SUC; 0.25 M) to produce six solutions for solid-surface vitrification. After warming, skin tissues were cultured in vitro and recovered cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity for developing the growth curve and determining the population doubling time (PDT), and viability by Trypan Blue. The vitrification did not alter the ability of the tissues to adhere to the culture dish, as well as the day of all explants with cell growth, subconfluence samples, subconfluence total time, and PDT (p > 0.05). Moreover, independent of the cryoprotectant solution used, the vitrification altered the day of all attached explants (p < 0.05). Nevertheless, for viability after the first passage, only the EG-SUC (86.9%) and DMSO-SUC (91.4%) groups maintained viable cell recovery similar to the nonvitrified group (96.3%, p > 0.05). Additionally, for viability after the third passage, only the EG-SUC group maintained the cell quality (88.3%), when compared with the nonvitrified (97.8%, p > 0.05). In conclusion, DMEM with 10% FBS, 3.0 M EG, and 0.25 M sucrose was the most efficient solution for vitrifying collared peccary skin tissues, leading to the in vitro culture of viable cells.

Resveratrol downregulates inflammatory pathway activated by lymphotoxin α (TNF-β) in articular chondrocytes: Comparison with TNF-α

PubMed Central

Buhrmann, Constanze; Popper, Bastian; Aggarwal, Bharat B.

2017-01-01

While Lymphotoxin α (TNF-β), a product of lymphocytes, is known to play a pivotal role in inflammatory joint environment, resveratrol has been shown to possess anti-inflammatory and chondroprotective effects via activation of the histondeacetylase Sirt1. Whether TNF-β induction of inflammatory pathways in primary human chondrocytes (PCH) can be modulated by resveratrol, was investigated. Monolayer and alginate cultures of PCH were treated with TNF-β, anti-TNF-β, nicotinamide (NAM), antisense oligonucleotides against Sirt1 (Sirt1-ASO) and/or resveratrol and co-cultured with T-lymphocytes. We found that resveratrol suppressed, similar to anti-TNF-β, TNF-β-induced increased adhesiveness in an inflammatory microenvironment of T-lymphocytes and PCH. In contrast, knockdown of Sirt1 by mRNA abolished the inhibitory effects of resveratrol on the TNF-β-induced adhesiveness, suggesting the essential role of this enzyme for resveratrol-mediated anti-inflammatory signaling. Similar results were obtained in PCH stimulated with TNF-α. Sirt1-ASO, NAM or TNF-β, similar to T-lymphocytes induced inflammatory microenvironment by down-regulation of cartilage-specific proteins, Sox9, Ki67 and enhanced NF-κB-regulated gene products involved in inflammatory and degradative processes in cartilage (MMP-9/-13, COX-2, caspase-3), NF-κB activation and its translocation to the nucleus. Moreover, resveratrol reversed the TNF-β-, NAM-, T-lymphocytes-induced up-regulation of various NF-κB-regulated gene products. Down-regulation of Sirt1 by mRNA interference abrogated the effect of resveratrol on TNF-β-induced effects. Ultrastructural and cell viability assay investigations revealed that resveratrol revoked TNF-β-induced dose-dependent degradative/apoptotic morphological changes, cell viability and proliferation in PCH. Taken together, suppression of TNF-β-induced inflammatory microenvironment in PCH by resveratrol/Sirt1 might be a novel therapeutic approach for targeting inflammation during rheumatoid arthritis. PMID:29095837

Heterogeneity of Focal Adhesions and Focal Contacts in Motile Fibroblasts.

PubMed

Gladkikh, Aleena; Kovaleva, Anastasia; Tvorogova, Anna; Vorobjev, Ivan A

2018-01-01

Cell-extracellular matrix (ECM) adhesion is an important property of virtually all cells in multicellular organisms. Cell-ECM adhesion studies, therefore, are very significant both for biology and medicine. Over the last three decades, biomedical studies resulted in a tremendous advance in our understanding of the molecular basis and functions of cell-ECM adhesion. Based on morphological and molecular criteria, several different types of model cell-ECM adhesion structures including focal adhesions, focal complexes, fibrillar adhesions, podosomes, and three-dimensional matrix adhesions have been described. All the subcellular structures that mediate cell-ECM adhesion are quite heterogeneous, often varying in size, shape, distribution, dynamics, and, to a certain extent, molecular constituents. The morphological "plasticity" of cell-ECM adhesion perhaps reflects the needs of cells to sense, adapt, and respond to a variety of extracellular environments. In addition, cell type (e.g., differentiation status, oncogenic transformation, etc.) often exerts marked influence on the structure of cell-ECM adhesions. Although molecular, genetic, biochemical, and structural studies provide important maps or "snapshots" of cell-ECM adhesions, the area of research that is equally valuable is to study the heterogeneity of FA subpopulations within cells. Recently time-lapse observations on the FA dynamics become feasible, and behavior of individual FA gives additional information on cell-ECM interactions. Here we describe a robust method of labeling of FA using plasmids with fluorescent markers for paxillin and vinculin and quantifying the morphological and dynamical parameters of FA.

Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion.

PubMed

Yamazaki, Daisuke; Oikawa, Tsukasa; Takenawa, Tadaomi

2007-01-01

During cadherin-dependent cell-cell adhesion, the actin cytoskeleton undergoes dynamic reorganization in epithelial cells. Rho-family small GTPases, which regulate actin dynamics, play pivotal roles in cadherin-dependent cell-cell adhesion; however, the precise molecular mechanisms that underlie cell-cell adhesion formation remain unclear. Here we show that Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE)-mediated reorganization of actin, downstream of Rac plays an important role in normal development of cadherin-dependent cell-cell adhesions in MDCK cells. Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization. The process of cell-cell adhesion is divided into three steps: formation of new cell-cell contacts, stabilization of these new contacts and junction maturation. WAVE1 and WAVE2 were expressed in MDCK cells. The functions of WAVE1 and WAVE2 were redundant in this system but WAVE2 appeared to play a more significant role. During the first step, WAVE2-dependent lamellipodial protrusions facilitated formation of cell-cell contacts. During the second step, WAVE2 recruited actin filaments to new cell-cell contacts and stabilized newly formed cadherin clusters. During the third step, WAVE2-dependent actin reorganization was required for organization and maintenance of mature cell-cell adhesions. Thus, Rac-WAVE-dependent actin reorganization is not only involved in formation of cell-cell adhesions but is also required for their maintenance.

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces.

PubMed

Nejidat, A; Saadi, I; Ronen, Z

2008-12-01

To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS-PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.

Microcontact printing of polydopamine on thermally expandable hydrogels for controlled cell adhesion and delivery of geometrically defined microtissues.

PubMed

Lee, Yu Bin; Kim, Se-Jeong; Kim, Eum Mi; Byun, Hayeon; Chang, Hyung-Kwan; Park, Jungyul; Choi, Yu Suk; Shin, Heungsoo

2017-10-01

Scaffold-free harvest of microtissue with a defined structure has received a great deal of interest in cell-based assay and regenerative medicine. In this study, we developed thermally expandable hydrogels with spatially controlled cell adhesive patterns for rapid harvest of geometrically controlled microtissue. We patterned polydopamine (PD) on to the hydrogel via microcontact printing (μCP), in linear shapes with widths of 50, 100 and 200μm. The hydrogels facilitated formation of spatially controlled strip-like microtissue of human dermal fibroblasts (HDFBs). It was possible to harvest and translocate microtissues with controlled widths of 61.4±14.7, 104.3±15.6, and 186.6±22.3μm from the hydrogel to glass substrates by conformal contact upon expansion of the hydrogel in response to a temperature change from 37 to 4°C, preserving high viability, extracellular matrix, and junction proteins. Microtissues were readily translocated in vivo to the subcutaneous tissue of mouse. The microtissues were further utilized as a simple assay model for monitoring of contraction in response to ROCK1 inhibitor. Collectively, micro-sized patterning of PD on the thermally expandable hydrogels via μCP holds promise for the development of microtissue harvesting systems that can be employed to ex vivo tissue assay and cell-based therapy. Harvest of artificial tissue with controlled cellular arrangement independently from external materials has been widely studied in cell-based assay and regenerative medicine. In this study, we developed scaffold-free harvest system of microtissues with anisotropic arrangement and controlled width by exploiting thermally expandable hydrogels with cell-adhesive patterns of polydopamine formed by simple microcontact printing. Cultured strips of human dermal fibroblasts on the hydrogels were rapidly delivered to various targets ranging from flat coverglass to mice subcutaneous tissue by thermal expansion of the hydrogel at 4°C for 10min. These were further utilized as a drug screening model responding to ROCK1 inhibitor, which imply its versatile applicability. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

PubMed

Shinohara, M; Kodama, A; Matozaki, T; Fukuhara, A; Tachibana, K; Nakanishi, H; Takai, Y

2001-06-01

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Changes in E-cadherin rigidity sensing regulate cell adhesion

PubMed Central

Collins, Caitlin; Pruitt, Beth L.; Nelson, W. James

2017-01-01

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion. PMID:28674019

Nanocellulose based asymmetric composite membrane for the multiple functions in cell encapsulation.

PubMed

Park, Minsung; Shin, Sungchul; Cheng, Jie; Hyun, Jinho

2017-02-20

We describe the nanocomposite membrane for cell encapsulation using nanocelluose hydrogels. One of the surfaces of bacterial cellulose (BC) pellicles was coated with collagen to enhance cell adhesion and the opposite side of the BC pellicles was coated with alginate to protect transplanted cells from immune rejection by the reduced pore size of the composite membrane. The morphology of nanocomposite membrane was observed by scanning electron microscopy and the permeability of the membrane was estimated by the release test using different molecular weights of polymer solution. The nanocomposite membrane was permeable to small molecules but impermeable to large molecules such as IgG antibodies inferring the potential use in cell implantation. In addition, the BC-based nanocomposite membrane showed a superior mechanical property due to the incorporation of compared with alginate membranes. The cells attached efficiently to the surface of BC composite membranes with a high level of cell viability as well as bioactivity. Cells grown on the BC composite membrane kit released dopamine freely to the medium through the membrane, which showed that the BC composite membrane would be a promising cell encapsulation material in implantation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gelatin- and starch-based hydrogels. Part B: In vitro mesenchymal stem cell behavior on the hydrogels.

PubMed

Van Nieuwenhove, Ine; Salamon, Achim; Adam, Stefanie; Dubruel, Peter; Van Vlierberghe, Sandra; Peters, Kirsten

2017-04-01

Tissue regeneration often occurs only to a limited extent. By providing a three-dimensional matrix serving as a surrogate extracellular matrix that promotes adult stem cell adhesion, proliferation and differentiation, scaffold-guided tissue regeneration aims at overcoming this limitation. In this study, we applied hydrogels made from crosslinkable gelatin, the hydrolyzed form of collagen, and functionalized starch which were characterized in depth and optimized as described in Van Nieuwenhove et al., 2016. "Gelatin- and Starch-Based Hydrogels. Part A: Hydrogel Development, Characterization and Coating", Carbohydrate Polymers 152:129-39. Collagen is the main structural protein in animal connective tissue and the most abundant protein in mammals. Starch is a carbohydrate consisting of a mixture of amylose and amylopectin. Hydrogels were developed with varying chemical composition (ratio of starch to gelatin applied) and different degrees of methacrylation of the applied gelatin phase. The hydrogels used exhibited no adverse effect on viability of the stem cells cultured on them. Moreover, initial cell adhesion did not differ significantly between them, while the strongest proliferation was observed on the hydrogel with the highest degree of cross-linking. On the least crosslinked and thus most flexible hydrogels, the highest degree of adipogenic differentiation was found, while osteogenic differentiation was the strongest on the most rigid, starch-blended hydrogels. Hydrogel coating with extracellular matrix compounds aggrecan or fibronectin prior to cell seeding exhibited no significant effects. Thus, gelatin-based hydrogels can be optimized regarding maximum promotion of either adipogenic or osteogenic stem cell differentiation in vitro, which makes them promising candidates for in vivo evaluation in clinical studies aiming at either soft or hard tissue regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles in human cardiac microvascular endothelial cells: cytotoxicity, permeability, and inflammation of metal oxide nanoparticles.

PubMed

Sun, Jing; Wang, Shaochuang; Zhao, Dong; Hun, Fei Han; Weng, Lei; Liu, Hui

2011-10-01

Wide applications and extreme potential of metal oxide nanoparticles (NPs) increase occupational and public exposure and may yield extraordinary hazards for human health. Exposure to NPs has a risk for dysfunction of the vascular endothelial cells. The objective of this study was to assess the cytotoxicity of six metal oxide NPs to human cardiac microvascular endothelial cells (HCMECs) in vitro. Metal oxide NPs used in this study included zinc oxide (ZnO), iron(III) oxide (Fe(2)O(3)), iron(II,III) oxide (Fe(3)O(4)), magnesium oxide (MgO), aluminum oxide (Al(2)O(3)), and copper(II) oxide (CuO). The cell viability, membrane leakage of lactate dehydrogenase, intracellular reactive oxygen species, permeability of plasma membrane, and expression of inflammatory markers vascular cell adhesion molecule-1, intercellular adhesion molecule-1, macrophage cationic peptide-1, and interleukin-8 in HCMECs were assessed under controlled and exposed conditions (12-24 h and 0.001-100 μg/ml of exposure). The results indicated that Fe(2)O(3), Fe(3)O(4), and Al(2)O(3) NPs did not have significant effects on cytotoxicity, permeability, and inflammation response in HCMECs at any of the concentrations tested. ZnO, CuO, and MgO NPs produced the cytotoxicity at the concentration-dependent and time-dependent manner, and elicited the permeability and inflammation response in HCMECs. These results demonstrated that cytotoxicity, permeability, and inflammation in vascular endothelial cells following exposure to metal oxide nanoparticles depended on particle composition, concentration, and exposure time. © Springer Science+Business Media B.V. 2011

Quantitative comparison of cancer and normal cell adhesion using organosilane monolayer templates: an experimental study on the anti-adhesion effect of green-tea catechins.

PubMed

Sakamoto, Rumi; Kakinuma, Eisuke; Masuda, Kentaro; Takeuchi, Yuko; Ito, Kosaku; Iketaki, Kentaro; Matsuzaki, Takahisa; Nakabayashi, Seiichiro; Yoshikawa, Hiroshi Y; Yamamoto, Hideaki; Sato, Yuko; Tanii, Takashi

2016-09-01

The main constituent of green tea, (-)-Epigallocatechin-3-O-gallate (EGCG), is known to have cancer-specific chemopreventive effects. In the present work, we investigated how EGCG suppresses cell adhesion by comparing the adhesion of human pancreatic cancer cells (AsPC-1 and BxPC-3) and their counterpart, normal human embryonic pancreas-derived cells (1C3D3), in catechin-containing media using organosilane monolayer templates (OMTs). The purpose of this work is (1) to evaluate the quantitativeness in the measurement of cell adhesion with the OMT and (2) to show how green-tea catechins suppress cell adhesion in a cancer-specific manner. For the first purpose, the adhesion of cancer and normal cells was compared using the OMT. The cell adhesion in different type of catechins such as EGCG, (-)-Epicatechin-3-O-gallate (ECG) and (-)-Epicatechin (EC) was also evaluated. The measurements revealed that the anti-adhesion effect of green-tea catechins is cancer-specific, and the order is EGCG≫ECG>EC. The results agree well with the data reported to date, showing the quantitativeness of the new method. For the second purpose, the contact area of cells on the OMT was measured by reflection interference contrast microscopy. The cell-OMT contact area of cancer cells decreases with increasing EGCG concentration, whereas that of normal cells remains constant. The results reveal a twofold action of EGCG on cancer cell adhesion-suppressing cell attachment to a candidate adhesion site and decreasing the contact area of the cells-and validates the use of OMT as a tool for screening cancer cell adhesion.

Characterization and human gingival fibroblasts biocompatibility of hydroxyapatite/PMMA nanocomposites for provisional dental implant restoration

NASA Astrophysics Data System (ADS)

Zhang, Jingchao; Liao, Juan; Mo, Anchun; Li, Yubao; Li, Jidong; Wang, Xuejiang

2008-11-01

The aim of this study was to determine nHA/PMMA composites (H/P) in an optimal ratio with improved cytocompatibility as well as valid physical properties for provisional dental implant restoration. 20 wt.%, 30 wt.%, 40 wt.% and 50 wt.% H/P were developed and characterized using XPS, bending strength test and SEM. Human gingival fibroblasts cultured in extracts or directly on sample discs were investigated by fluorescent staining and MTT assay. Chemical integration in nHA/PMMA interface was indicated by XPS. Typical fusiform cells with adhesion spots were detected on H/P discs. MTT results also indicated higher cell viability in 30 wt.% and 40 wt.% H/P discs ( P < 0.05). We conclude that nHA addition to PMMA enhances cytocompatibility and the optimal nHA/PMMA ratio for provisional fixed crowns (PFC) is 0.4:1.

Combining Dynamic Stretch and Tunable Stiffness to Probe Cell Mechanobiology In Vitro

PubMed Central

Throm Quinlan, Angela M.; Sierad, Leslie N.; Capulli, Andrew K.; Firstenberg, Laura E.; Billiar, Kristen L.

2011-01-01

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch. PMID:21858051

Hormonal Regulation and Distinct Functions of Semaphorin-3B and Semaphorin-3F in Ovarian Cancer

PubMed Central

Joseph, Doina; Ho, Shuk-Mei; Syed, Viqar

2009-01-01

Semaphorins comprise a family of molecules that influence neuronal growth and guidance. Class-3 semaphorins, semaphorin-3B (SEMA3B) and semaphorin-3F (SEMA3F) illustrate their effects by forming a complex with neuropilins (NP-1 or NP-2) and plexins. We examined the status and regulation of semaphorins and their receptors in human ovarian cancer cells. A significantly reduced expression of SEMA3B (83 kD), SEMA3F (90 kD), and plexin-A3 was observed in ovarian cancer (OVCA) cell lines when compared to normal human ovarian surface epithelial (HOSE) cells. The expression of NP-1, NP-2 and plexin-A1 was not altered in HOSE and OVCA cells. The decreased expression of SEMA3B, SEMA3F, and plexin-A3 was confirmed in stage 3 ovarian tumors. Treatment of OVCA cells with luteinizing hormone, follicle-stimulating hormone, and estrogen induced a significant upregulation of SEMA3B, whereas SEMA3F was upregulated only by estrogen. Co-treatment of cell lines with a hormone and its specific antagonist blocked the effect of the hormone. Ectopic expression of SEMA3B or SEMA3F reduced soft-agar colony formation, adhesion, and cell invasion of OVCA cell cultures. Forced expression of SEMA3B, but not SEMA3F, inhibited viability of OVCA cells. Overexpression of SEMA3B and SEMA3F reduced focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase (MMP)-2 and -9 expression in OVCA cells. Forced expression of SEMA3F, but not SEMA3B in OVCA cells, significantly inhibited endothelial cell tube formation. Collectively, our results suggest loss of SEMA3 expression could be a hallmark of cancer progression. Furthermore, gonadotropin- and/or estrogen-mediated maintenance of SEMA3 expression could control ovarian cancer angiogenesis and metastasis. PMID:20124444

Electrospun Polyhydroxybutyrate/Poly(ε-caprolactone)/58S Sol-Gel Bioactive Glass Hybrid Scaffolds with Highly Improved Osteogenic Potential for Bone Tissue Engineering.

PubMed

Ding, Yaping; Li, Wei; Müller, Teresa; Schubert, Dirk W; Boccaccini, Aldo R; Yao, Qingqing; Roether, Judith A

2016-07-13

Electrospinning of biopolymer and inorganic substances is one of the efficient ways to combine various advantageous properties in one single fibrous structure with potential for tissue engineering applications. In the present study, to integrate the high stiffness of polyhydroxybutyrate (PHB), the flexibility of poly(ε-caprolactone) (PCL) and the bioactivity of 58S bioactive glass, PHB/PCL/58S sol-gel bioactive glass hybrid scaffolds were fabricated using combined electrospinning and sol-gel method. Physical features such as fiber diameter distribution, mechanical strength and Young's modulus were characterized thoroughly. FTIR analysis demonstrated the successful incorporation of 58S bioactive glass into the blend polymers, which greatly improved the hydrophilicity of PHB/PCL fibermats. The primary biological response of MG-63 osteoblast-like cells on the prepared fibrous scaffolds was evaluated, proving that the 58S glass sol containing hybrid scaffold were not only favorable to MG-63 cell adhesion but also slightly enhanced cell viability and significantly increased alkaline phosphate activity .

Antimicrobial polycaprolactone/polyethylene glycol embedded lysozyme coatings of Ti implants for osteoblast functional properties in tissue engineering

NASA Astrophysics Data System (ADS)

Visan, A.; Cristescu, R.; Stefan, N.; Miroiu, M.; Nita, C.; Socol, M.; Florica, C.; Rasoga, O.; Zgura, I.; Sima, L. E.; Chiritoiu, M.; Chifiriuc, M. C.; Holban, A. M.; Mihailescu, I. N.; Socol, G.

2017-09-01

In this study, coatings based on lysozyme embedded into a matrix of polyethylene glycol (PEG) and polycaprolactone (PCL) were fabricated by two different methods (Matrix Assisted Pulsed Laser Evaporation - MAPLE and Dip Coating) for obtaining antimicrobial coatings envisaged for long term medical applications. Coatings with different PEG:PCL compositions (3:1; 1:1; 1:3) were synthesized in order to evaluate the antimicrobial activity of lysozyme embedded into the polymeric matrix. The main surface features, such as roughness and wettability, with impact on the microbial adhesion as well as on the eukaryote cell function were measured. The obtained composite coatings exhibited a significant antibacterial activity against Escherichia coli, Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus strains. As well, specific blended coatings showed appropriate viability, good spreading and normal cell morphology of SaOs2 human osteoblasts and mesenchymal stem cells (MSCs). These investigations highlight the suitability of biodegradable composites as implant coatings for decreasing the risk of bacterial contamination associated with prosthetic procedures.

Endothelialization of TiO2 Nanorods Coated with Ultrathin Amorphous Carbon Films

NASA Astrophysics Data System (ADS)

Chen, Hongpeng; Tang, Nan; Chen, Min; Chen, Dihu

2016-03-01

Carbon plasma nanocoatings with controlled fraction of sp3-C bonding were deposited on TiO2 nanorod arrays (TNAs) by DC magnetic-filtered cathodic vacuum arc deposition (FCVAD). The cytocompatibility of TNA/carbon nanocomposites was systematically investigated. Human umbilical vein endothelial cells (HUVECs) were cultured on the nanocomposites for 4, 24, and 72 h in vitro. It was found that plasma-treated TNAs exhibited excellent cell viability as compared to the untreated. Importantly, our results show that cellular responses positively correlate with the sp3-C content. The cells cultured on high sp3-C-contented substrates exhibit better attachment, shape configuration, and proliferation. These findings indicate that the nanocomposites with high sp3-C content possessed superior cytocompatibility. Notably, the nanocomposites drastically reduced platelet adhesion and activation in our previous studies. Taken together, these findings suggest the TNA/carbon scaffold may serve as a guide for the design of multi-functionality devices that promotes endothelialization and improves hemocompatibility.

PAK4 kinase is essential for embryonic viability and for proper neuronal development.

PubMed

Qu, Jian; Li, Xiaofan; Novitch, Bennet G; Zheng, Ye; Kohn, Matthew; Xie, Jian-Ming; Kozinn, Spencer; Bronson, Roderick; Beg, Amer A; Minden, Audrey

2003-10-01

The serine/threonine kinase PAK4 is a target for the Rho GTPase Cdc42 and has been shown to regulate cell morphology and cytoskeletal organization in mammalian cells. To examine the physiological and developmental functions of PAK4, we have disrupted the PAK4 gene in mice. The absence of PAK4 led to lethality by embryonic day 11.5, a result most likely due to a defect in the fetal heart. Striking abnormalities were also evident in the nervous systems of PAK4-deficient embryos. These embryos had dramatic defects in neuronal development and axonal outgrowth. In particular, spinal cord motor neurons and interneurons failed to differentiate and migrate to their proper positions. This is probably related to the role for PAK4 in the regulation of cytoskeletal organization and cell and/or extracellular matrix adhesion. PAK4-null embryos also had defects in proper folding of the caudal portion of the neural tube, suggesting an important role for PAK4 in neural tube development.

Differentiation of bone marrow stromal cells in poly(lactide-co-glycolide)/chitosan scaffolds.

PubMed

Kuo, Yung-Chih; Yeh, Chun-Fu; Yang, Jen-Tsung

2009-12-01

This study investigates the physicochemical properties of poly(lactide-co-glycolide) (PLGA)/chitosan scaffolds and the neuron growth factor (NGF)-guided differentiation of bone marrow stromal cells (BMSCs) in the scaffolds. The scaffolds were prepared by the crosslinking of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and genipin, and the differentiating BMSCs were characterized against CD44, CD90 and NeuN. The scaffold with 20% PLGA yielded 95% porosity, Young's modulus of 13MPa, 70% adhesion of BMSCs and 1.6-fold increase in the cell viability over 7-day cultivation. BMSCs without guidance in the PLGA/chitosan scaffolds were prone to differentiate toward osteoblasts with apparent deposition of calcium. When NGF was introduced, an increased weight percentage of PLGA yielded more identified neurons. In addition, mature neurons emerged from the PLGA-rich biomaterials after induction with NGF over 2 days. A proper control over the physical and biomedical property of the scaffolds and the NGF-guided differentiation of BMSCs can be promising for nerve regeneration.

Non-thermal atmospheric-pressure plasma possible application in wound healing.

PubMed

Haertel, Beate; von Woedtke, Thomas; Weltmann, Klaus-Dieter; Lindequist, Ulrike

2014-11-01

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.

Chitosan-hyaluronan/nano chondroitin sulfate ternary composite sponges for medical use.

PubMed

Anisha, B S; Sankar, Deepthi; Mohandas, Annapoorna; Chennazhi, K P; Nair, Shantikumar V; Jayakumar, R

2013-02-15

In this work chitosan-hyaluronan composite sponge incorporated with chondroitin sulfate nanoparticle (nCS) was developed. The fabrication of hydrogel was based on simple ionic cross-linking using EDC, followed by lyophilization to obtain the composite sponge. nCS suspension was characterized using DLS and SEM and showed a size range of 100-150 nm. The composite sponges were characterized using SEM, FT-IR and TG-DTA. Porosity, swelling, biodegradation, blood clotting and platelet activation of the prepared sponges were also evaluated. Nanocomposites showed a porosity of 67% and showed enhanced swelling and blood clotting ability. Cytocompatibility and cell adhesion studies of the sponges were done using human dermal fibroblast (HDF) cells and the nanocomposite sponges showed more than 90% viability. Nanocomposite sponges also showed enhanced proliferation of HDF cells within two days of study. These results indicated that this nanocomposite sponges would be a potential candidate for wound dressing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Non-Thermal Atmospheric-Pressure Plasma Possible Application in Wound Healing

PubMed Central

Haertel, Beate; von Woedtke, Thomas; Weltmann, Klaus-Dieter; Lindequist, Ulrike

2014-01-01

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out. PMID:25489414

An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

PubMed Central

Collins, Caitlin

2014-01-01

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion. PMID:25452388

Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces – A Novel Method of Enhancing Flow-Resistant Cell Adhesion

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; McCracken, Katherine E.; Riley, Mark R.

2014-01-01

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear. PMID:23225491

In vitro corrosion and biocompatibility of binary magnesium alloys.

PubMed

Gu, Xuenan; Zheng, Yufeng; Cheng, Yan; Zhong, Shengping; Xi, Tingfei

2009-02-01

As bioabsorbable materials, magnesium alloys are expected to be totally degraded in the body and their biocorrosion products not deleterious to the surrounding tissues. It's critical that the alloying elements are carefully selected in consideration of their cytotoxicity and hemocompatibility. In the present study, nine alloying elements Al, Ag, In, Mn, Si, Sn, Y, Zn and Zr were added into magnesium individually to fabricate binary Mg-1X (wt.%) alloys. Pure magnesium was used as control. Their mechanical properties, corrosion properties and in vitro biocompatibilities (cytotoxicity and hemocompatibility) were evaluated by SEM, XRD, tensile test, immersion test, electrochemical corrosion test, cell culture and platelet adhesion test. The results showed that the addition of alloying elements could influence the strength and corrosion resistance of Mg. The cytotoxicity tests indicated that Mg-1Al, Mg-1Sn and Mg-1Zn alloy extracts showed no significant reduced cell viability to fibroblasts (L-929 and NIH3T3) and osteoblasts (MC3T3-E1); Mg-1Al and Mg-1Zn alloy extracts indicated no negative effect on viabilities of blood vessel related cells, ECV304 and VSMC. It was found that hemolysis and the amount of adhered platelets decreased after alloying for all Mg-1X alloys as compared to the pure magnesium control. The relationship between the corrosion products and the in vitro biocompatibility had been discussed and the suitable alloying elements for the biomedical applications associated with bone and blood vessel had been proposed.

Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels

PubMed Central

Thibault, Richard; Ambrose, Winnette McIntosh; Schein, Oliver D.; Chakravarti, Shukti; Elisseeff, Jennifer

2015-01-01

The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase–polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment. PMID:18567550

Influence of Cu-Ti thin film surface properties on antimicrobial activity and viability of living cells.

PubMed

Wojcieszak, Damian; Kaczmarek, Danuta; Antosiak, Aleksandra; Mazur, Michal; Rybak, Zbigniew; Rusak, Agnieszka; Osekowska, Malgorzata; Poniedzialek, Agata; Gamian, Andrzej; Szponar, Bogumila

2015-11-01

The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown. Copyright © 2015 Elsevier B.V. All rights reserved.

Nano-hydroxyapatite/β-CD/chitosan nanocomposite for potential applications in bone tissue engineering.

PubMed

Shakir, Mohammad; Jolly, Reshma; Khan, Mohd Shoeb; Rauf, Ahmar; Kazmi, Shadab

2016-12-01

Herein, we report the synthesis of a novel tri-component nanocomposite system incorporating β-cyclodextrin (β-CD) with nano-hydroxyapatite (n-HA) and chitosan (CS), (n-HA/β-CD/CS) at three different temperatures via co-precipitation method. The chemical interactions and surface morphology have been evaluated by TEM, SEM and AFM techniques revealing the agglomerated nanoparticles in CS/n-HA-HA binary system whereas the ternary systems produced needle shaped nanoparticles dispersed homogeneously at low temperature with more porous and rougher surface. The addition of β-CD in CS/n-HA at low temperature decreased the particle size and raised the thermal stability as compared to CS/n-HA. The comparative hemolytic, protein adsorption and platelet adhesion studies confirmed the better hemocompatibility of n-HA/β-CD/CS-(RT,HT,LT) nanocomposites relative to CS/n-HA. The cell viability has been evaluated in vitro using MG-63 cell line which revealed superior non toxicity of n-HA/β-CD/CS-LT nanocomposite in comparison to n-HA/β-CD/CS-(RT,HT) and CS/n-HA nanocomposites. Thus it may be concluded that the orchestrated organic/inorganic n-HA/β-CD/CS-(RT,HT,LT) nanocomposites exhibited relatively higher cell viability of human osteoblast cells, stimulated greater osteogenesis, controlled biodegradation, enhanced antibacterial activity with excellent in-vitro biomineralization and remarkable mechanical parameters as compared to CS/n-HA nanocomposite and thus may provide opportunities for potential use as an alternative biomaterial for Bone tissue engineering applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Modulating the Release Kinetics of Paclitaxel from Membrane-Covered Stents Using Different Loading Strategies

PubMed Central

Sydow-Plum, Georg; Haidar, Ziyad S.; Merhi, Yahye; Tabrizian, Maryam

2008-01-01

Membrane-covered Express2TM Monorail® stents composed of chitosan (CH) blended with polyethylene oxide (PEO) in 70:30% wt (CH-PEO) were coated with a monolayer of hyaluronic acid (HA). This significantly improved the resistance to platelet adhesion and demonstrated excellent mechanical properties, resisting the harsh conditions during stent crimping and subsequent inflation. CH-PEO/HA membrane was then combined with a paclitaxel (Pac) delivery system via three different approaches for comparison of release profiles of Pac. The activity of Pac in these systems was confirmed since its presence in the membrane significantly decreased cell viability of U937 macrophages. Presented results are promising for applications requiring different release patterns of hydrophobic drugs.

Antimetastatic Effects of Phyllanthus on Human Lung (A549) and Breast (MCF-7) Cancer Cell Lines

PubMed Central

Lee, Sau Har; Jaganath, Indu Bala; Wang, Seok Mui; Sekaran, Shamala Devi

2011-01-01

Background Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells. Methodology/Principal Findings Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC50 values ranging from 50–180 µg/ml and 65–470 µg/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 µg/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20–200 µg/ml for methanolic extracts and 50–500 µg/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts. Conclusions/Significance The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers. PMID:21698198

Effect of the Streptococcus agalactiae Virulence Regulator CovR on the Pathogenesis of Urinary Tract Infection.

PubMed

Sullivan, Matthew J; Leclercq, Sophie Y; Ipe, Deepak S; Carey, Alison J; Smith, Joshua P; Voller, Nathan; Cripps, Allan W; Ulett, Glen C

2017-02-01

Streptococcus agalactiae can cause urinary tract infection (UTI). The role of the S. agalactiae global virulence regulator, CovR, in UTI pathogenesis is unknown. We used murine and human bladder uroepithelial cell models of UTI and S. agalactiae mutants in covR and related factors, including β-hemolysin/cytolysin (β-h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI. We found that covR-deficient serotype III S. agalactiae 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with covR-deficient S. agalactiae produced less proinflammatory cytokines than those infected with wild-type 874391. Acute cytotoxicity in uroepithelial cells triggered by covR-deficient but not wild-type 874391 was associated with significant caspase 3 activation. Mechanistically, covR mutation significantly altered the expression of several genes in S. agalactiae 874391 that encode key virulence factors, including β-h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the β-h/c, exerted significant effects on colonization of the murine urinary tract in vivo. S. agalactiae CovR promotes bladder infection and inflammation, as well as adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial, and influenced by virulence effects of CovR, HvgA, and capsule. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

PubMed

Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

2016-04-14

Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

Electroactive polymer-peptide conjugates for adhesive biointerfaces.

PubMed

Maione, Silvana; Gil, Ana M; Fabregat, Georgina; Del Valle, Luis J; Triguero, Jordi; Laurent, Adele; Jacquemin, Denis; Estrany, Francesc; Jiménez, Ana I; Zanuy, David; Cativiela, Carlos; Alemán, Carlos

2015-10-15

Electroactive polymer-peptide conjugates have been synthesized by combining poly(3,4-ethylenedioxythiophene), a polythiophene derivative with outstanding properties, and an Arg-Gly-Asp (RGD)-based peptide in which Gly has been replaced by an exotic amino acid bearing a 3,4-ethylenedioxythiophene ring in the side chain. The incorporation of the peptide at the ends of preformed PEDOT chains has been corroborated by both FTIR and X-ray photoelectron spectroscopy. Although the morphology and topology are not influenced by the incorporation of the peptide at the ends of PEDOT chains, this process largely affects other surface properties. Thus, the wettability of the conjugates is considerably higher than that of PEDOT, independently of the synthetic strategy, whereas the surface roughness only increases when the conjugate is obtained using a competing strategy (i.e. growth of the polymer chains against termination by end capping). The electrochemical activity of the conjugates has been found to be higher than that of PEDOT, evidencing the success of the polymer-peptide links designed by chemical similarity. Density functional theory calculations have been used not only to ascertain the conformational preferences of the peptide but also to interpret the electronic transitions detected by UV-vis spectroscopy. Electroactive surfaces prepared using the conjugates displayed the higher bioactivities in terms of cell adhesion, with the relative viabilities being dependent on the roughness, wettability and electrochemical activity of the conjugate. In addition to the influence of the peptide fragment in the initial cell attachment and subsequent cell spreading and survival, the results indicate that PEDOT promotes the exchange of ions at the conjugate-cell interface.

An Inducible Endothelial Cell Surface Glycoprotein Mediates Melanoma Adhesion

NASA Astrophysics Data System (ADS)

Rice, G. Edgar; Bevilacqua, Michael P.

1989-12-01

Hematogenous metastasis requires the arrest and extravasation of blood-borne tumor cells, possibly involving direct adhesive interactions with vascular endothelium. Cytokine activation of cultured human endothelium increases adhesion of melanoma and carcinoma cell lines. An inducible 110-kD endothelial cell surface glycoprotein, designated INCAM-110, appears to mediate adhesion of melanoma cells. In addition, an inducible endothelial receptor for neutrophils, ELAM-1, supports the adhesion of a human colon carcinoma cell line. Thus, activation of vascular endothelium in vivo that results in increased expression of INCAM-110 and ELAM-1 may promote tumor cell adhesion and affect the incidence and distribution of metastases.

9-cis-Retinoic Acid Promotes Cell Adhesion Through Integrin Dependent and Independent Mechanisms Across Immune Lineages

PubMed Central

Whelan, Jarrett T.; Chen, Jianming; Miller, Jabin; Morrow, Rebekah L.; Lingo, Joshuah D.; Merrell, Kaitlin; Shaikh, Saame Raza; Bridges, Lance C.

2012-01-01

Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) on human hematopoietic cell adhesion was investigated. 9-cis-RA treatment specifically induced cell adhesion of the human immune cell lines HuT-78, NB4, RPMI 8866, and U937. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if cell adhesion was integrin-dependent. By employing a variety of integrin antagonist including function-blocking antibodies and EDTA, we establish that 9-cis-RA prompts immune cell adhesion through established integrin receptors in addition to a novel integrin-independent process. The novel integrin-independent adhesion required the presence of retinoid and was attenuated by treatment with synthetic corticosteroids. Finally, we demonstrate that 9-cis-RA treatment of primary murine B-cells induces ex vivo adhesion that persists in the absence of integrin function. Our study is the first to demonstrate that 9-cis-retinoic acid influences immune cell adhesion through at least two functionally distinct mechanisms. PMID:22925918

Role of cellular adhesions in tissue dynamics spectroscopy

NASA Astrophysics Data System (ADS)

Merrill, Daniel A.; An, Ran; Turek, John; Nolte, David

2014-02-01

Cellular adhesions play a critical role in cell behavior, and modified expression of cellular adhesion compounds has been linked to various cancers. We tested the role of cellular adhesions in drug response by studying three cellular culture models: three-dimensional tumor spheroids with well-developed cellular adhesions and extracellular matrix (ECM), dense three-dimensional cell pellets with moderate numbers of adhesions, and dilute three-dimensional cell suspensions in agarose having few adhesions. Our technique for measuring the drug response for the spheroids and cell pellets was biodynamic imaging (BDI), and for the suspensions was quasi-elastic light scattering (QELS). We tested several cytoskeletal chemotherapeutic drugs (nocodazole, cytochalasin-D, paclitaxel, and colchicine) on three cancer cell lines chosen from human colorectal adenocarcinoma (HT-29), human pancreatic carcinoma (MIA PaCa-2), and rat osteosarcoma (UMR-106) to exhibit differences in adhesion strength. Comparing tumor spheroid behavior to that of cell suspensions showed shifts in the spectral motion of the cancer tissues that match predictions based on different degrees of cell-cell contacts. The HT-29 cell line, which has the strongest adhesions in the spheroid model, exhibits anomalous behavior in some cases. These results highlight the importance of using three-dimensional tissue models in drug screening with cellular adhesions being a contributory factor in phenotypic differences between the drug responses of tissue and cells.

Silicon Carbide Nanoparticles as an Effective Bioadhesive to Bond Collagen Containing Composite Gel Layers for Tissue Engineering Applications.

PubMed

Attalla, Rana; Ling, Celine S N; Selvaganapathy, Ponnambalam Ravi

2018-03-01

Additive manufacturing via layer-by-layer adhesive bonding holds much promise for scalable manufacturing of tissue-like constructs, specifically scaffolds with integrated vascular networks for tissue engineering applications. However, there remains a lack of effective adhesives capable of composite layer fusion without affecting the integrity of patterned features. Here, the use of silicon carbide is introduced as an effective adhesive to achieve strong bonding (0.39 ± 0.03 kPa) between hybrid hydrogel films composed of alginate and collagen. The techniques have allowed us to fabricate multilayered, heterogeneous constructs with embedded high-resolution microchannels (150 µm-1 mm) that are precisely interspaced (500-600 µm). Hydrogel layers are effectively bonded with silicon carbide nanoparticles without blocking the hollow microchannels and high cell viability (90.61 ± 3.28%) is maintained within the scaffold. Nanosilica is also tested and found to cause clogging of smaller microchannels when used for interlayer bonding, but is successfully used to attach synthetic polymers (e.g., Tygon) to the hydrogels (32.5 ± 2.12 mN bond strength). This allows us to form inlet and outlet interconnections to the gel constructs. This ability to integrate hollow channel networks into bulk soft material structures for perfusion can be useful in 3D tissue engineering applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of Neutrophil Adhesion and Antimicrobial Activity by Diluted Hydrosol Prepared from Rosa damascena.

PubMed

Maruyama, Naho; Tansho-Nagakawa, Shigeru; Miyazaki, Chizuru; Shimomura, Kazuyuki; Ono, Yasuo; Abe, Shigeru

2017-01-01

Hydrosol prepared from the flowers of Rosa damascena (rose water) has been traditionally used for various health-related issues, including skin troubles such as erythema, itchiness, swelling. For the care of these skin troubles caused by microbial infection, both antimicrobial and antiinflammatory effects are required. Here, we investigated the effects of rose water on the growth of Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), which cause skin infections, and on the function of neutrophils, which play a major role in the regulation of inflammatory reactions. To assess its modulatory effects on neutrophils, the effects of rose water against neutrophil adhesion response were evaluated. Rose water inhibited mycelial growth of C. albicans at a concentration of ca. 2.2%, and reduced viability of MRSA within 1 h. Rose water suppressed neutrophil activation induced by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and N-formyl-Met-Leu-Phe (fMLP) at 5-15%. It also reduced the LPS- and TNF-α-induced cell surface expression of the adhesion-related molecule, cluster of differentiation (CD) 11b, but did not affect the migratory capacity of neutrophils with or without chemoattractant. These results suggest that rose water may reduce the pathogenicity of microbes, and attenuate neutrophil stimulation, which is involved in inflammatory responses. These findings suggest that rose water has a potential effect to inhibit skin inflammation caused by microbes.

Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.

PubMed

Voss, Alexandra; Wei, HongYing; Zhang, Yi; Turner, Stuart; Ceccone, Giacomo; Reithmaier, Johann Peter; Stengl, Monika; Popov, Cyril

2016-07-01

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of Human Denuded Amniotic Membrane and Porcine Small Intestine Submucosa as Scaffolds for Limbal Mesenchymal Stem Cells.

PubMed

Sous Naasani, Liliana I; Rodrigues, Cristiano; Azevedo, Jéssica Gonçalves; Damo Souza, Aline F; Buchner, Silvio; Wink, Márcia R

2018-04-29

Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.

Photochemical bonding of epithelial cell-seeded collagen lattice to rat muscle layer for esophageal tissue engineering: a pilot study

NASA Astrophysics Data System (ADS)

Chan, Barbara P.; Sato, M.; Vacanti, Joseph P.; Kochevar, Irene E.; Redmond, Robert W.

2005-04-01

Bilayered tube structures consist of epithelial cell-seeded collagen lattice and muscle layer have been fabricated for esophageal tissue engineering. Good adhesion between layers in order to facilitate cell infiltration and neovascularization in the collagen lattice is required. Previous efforts include using other bioglues such as fibrin glue and silicone tube as the physical support. However, the former is subjected to chances of transmitting blood-born infectious disease and is time consuming while the latter requires a second surgical procedure. The current project aimed to bond the cell-seeded collagen lattice to muscle layer using photochemical bonding, which has previously been demonstrated a rapid and non-thermal procedure in bonding collagenous tissues. Rat esophageal epithelial cells were seeded on collagen lattice and together with the latissimus dorsi muscle layer, were exposed to a photosensitizer rose Bengal at the bonding surface. An argon laser was used to irradiate the approximated layers. Bonding strength was measured during the peeling test of the collagen layer from the muscle layer. Post-bonding cell viability was assessed using a modified NADH-diaphorase microassay. A pilot in vivo study was conducted by directly bonding the cell-seeded collagen layer onto the muscle flap in rats and the structures were characterized histologically. Photochemical bonding was found to significantly increase the adherence at the bonding interface without compromising the cell viability. This indicates the feasibility of using the technique to fabricate multi-layered structures in the presence of living cells. The pilot animal study demonstrated integration of the collagen lattice with the muscle layer at the bonding interface although the subsequent surgical manipulation disturbed the integration at some region. This means that an additional procedure removing the tube could be avoided if the approximation and thus the bonding are optimized. Cell infiltration and neovascularization were also evident demonstrating that direct bonding of engineered tissue structures in particular those with low processability such as collagen lattice to the host tissue is feasible.

Holographic lithography for biomedical applications

NASA Astrophysics Data System (ADS)

Stankevicius, E.; Balciunas, E.; Malinauskas, M.; Raciukaitis, G.; Baltriukiene, D.; Bukelskiene, V.

2012-06-01

Fabrication of scaffolds for cell growth with appropriate mechanical characteristics is top-most important for successful creation of tissue. Due to ability of fast fabrication of periodic structures with a different period, the holographic lithography technique is a suitable tool for scaffolds fabrication. The scaffolds fabricated by holographic lithography can be used in various biomedical investigations such as the cellular adhesion, proliferation and viability. These investigations allow selection of the suitable material and geometry of scaffolds which can be used in creation of tissue. Scaffolds fabricated from di-acrylated poly(ethylene glycol) (PEG-DA-258) over a large area by holographic lithography technique are presented in this paper. The PEG-DA scaffolds fabricated by holographic lithography showed good cytocompatibility for rabbit myogenic stem cells. It was observed that adult rabbit muscle-derived myogenic stem cells grew onto PEG-DA scaffolds. They were attached to the pillars and formed cell-cell interactions. It demonstrates that the fabricated structures have potential to be an interconnection channel network for cell-to-cell interactions, flow transport of nutrients and metabolic waste as well as vascular capillary ingrowth. These results are encouraging for further development of holographic lithography by improving its efficiency for microstructuring three-dimensional scaffolds out of biodegradable hydrogels

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

PubMed

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

2016-09-14

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

In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits.

PubMed

Keilhoff, G; Fansa, H; Schneider, W; Wolf, G

1999-07-01

In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation. The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps. The rat sciatic nerve used as a model was transected distally to the spinal ganglia. Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks. The nerve was then resected and prepared for cell cultivation. Schwann cells cultivated from the contralateral untreated nerve served as control. Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures. Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation. The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased. Best results were obtained 1 week after predegeneration.

Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein.

PubMed

Lengerer, Birgit; Pjeta, Robert; Wunderer, Julia; Rodrigues, Marcelo; Arbore, Roberto; Schärer, Lukas; Berezikov, Eugene; Hess, Michael W; Pfaller, Kristian; Egger, Bernhard; Obwegeser, Sabrina; Salvenmoser, Willi; Ladurner, Peter

2014-02-12

Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process. In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin. Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

PubMed

Palchesko, Rachelle N; Szymanski, John M; Sahu, Amrita; Feinberg, Adam W

2014-09-01

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo . Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment

PubMed Central

Palchesko, Rachelle N.; Szymanski, John M.; Sahu, Amrita; Feinberg, Adam W.

2014-01-01

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments. PMID:25530816

Influence of non-thermal TiCl4/Ar+O2 plasma-assisted TiOx based coatings on the surface of polypropylene (PP) films for the tailoring of surface properties and cytocompatibility.

PubMed

Pandiyaraj, K N; Kumar, A Arun; Ramkumar, M C; Sachdev, A; Gopinath, P; Cools, Pieter; De Geyter, N; Morent, R; Deshmukh, R R; Hegde, P; Han, C; Nadagouda, M N

2016-05-01

The superior bulk properties (corrosion resistance, high strength to weight ratio, relatively low cost and easy processing) of hydrocarbon based polymers such as polypropylene (PP) have contributed significantly to the development of new biomedical applications such as artificial organs and cell scaffolds. However, low cell affinity is one of the main draw backs for PP due to its poor surface properties. In tissue engineering, physico-chemical surface properties such as hydrophilicity, polar functional groups, surface charge and morphology play a crucial role to enrich the cell proliferation and adhesion. In this present investigation TiOx based biocompatible coatings were developed on the surface of PP films via DC excited glow discharge plasma, using TiCl4/Ar+O2 gas mixture as a precursor. Various TiOx-based coatings are deposited on the surface of PP films as a function of discharge power. The changes in hydrophilicity of the TiOx/PP film surfaces were studied using contact angle analysis and surface energy calculations by Fowke's approximation. X-ray photo-electron spectroscopy (XPS) was used to investigate the surface chemical composition of TiOx/PP films. The surface morphology of the obtained TiOx/PP films was investigated by scanning electron and transmission electron microscopy (SEM &TEM). Moreover, the surface topography of the material was analyzed by atomic force microscopy (AFM). The cytocompatibility of the TiOx/PP films was investigated via in vitro analysis (cell viability, adhesion and cytotoxicity) using NIH3T3 (mouse embryonic fibroblast) cells. Furthermore the antibacterial activities of TiOx/PP films were also evaluated against two distinct bacterial models namely Gram positive Staphylococcus aureus (S.aureus) and Gram negative Escherichia coli DH5α. (E.coli) bacteria. XPS results clearly indicate the successful incorporation of TiOx and oxygen containing polar functional groups on the surface of plasma treated PP films. Moreover the surface of modified PP films exhibited nano structured morphology, as confirmed by SEM, TEM and AFM. The physico-chemical changes have improved the hydrophilicity of the PP films. The in-vitro analysis clearly confirms that the TiOx coated PP films performs as good as the standard tissue culture plates and also are unlikely to impact the bacterial cell viability. Copyright © 2016 Elsevier B.V. All rights reserved.

[Expression of cell adhesion molecules in acute leukemia cell].

PubMed

Ju, Xiaoping; Peng, Min; Xu, Xiaoping; Lu, Shuqing; Li, Yao; Ying, Kang; Xie, Yi; Mao, Yumin; Xia, Fang

2002-11-01

To investigate the role of cell adhesion molecule in the development and extramedullary infiltration (EI) of acute leukemia. The expressions of neural cell adhesion molecule (NCAM) gene, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) genes in 25 acute leukemia patients bone marrow cells were detected by microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). The expressions of NCAM, ICAM-1 and VCAM-1 gene were significantly higher in acute leukemia cells and leukemia cells with EI than in normal tissues and leukemia cells without EI, respectively, both by cDNA microarray and by RT-PCR. The cDNA microarray is a powerful technique in analysis of acute leukemia cells associated genes. High expressions of cell adhesion molecule genes might be correlated with leukemia pathogenesis and infiltration of acute leukemia cell.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

PubMed

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

The cancer cell adhesion resistome: mechanisms, targeting and translational approaches.

PubMed

Dickreuter, Ellen; Cordes, Nils

2017-06-27

Cell adhesion-mediated resistance limits the success of cancer therapies and is a great obstacle to overcome in the clinic. Since the 1990s, where it became clear that adhesion of tumor cells to the extracellular matrix is an important mediator of therapy resistance, a lot of work has been conducted to understand the fundamental underlying mechanisms and two paradigms were deduced: cell adhesion-mediated radioresistance (CAM-RR) and cell adhesion-mediated drug resistance (CAM-DR). Preclinical work has evidently demonstrated that targeting of integrins, adapter proteins and associated kinases comprising the cell adhesion resistome is a promising strategy to sensitize cancer cells to both radiotherapy and chemotherapy. Moreover, the cell adhesion resistome fundamentally contributes to adaptation mechanisms induced by radiochemotherapy as well as molecular drugs to secure a balanced homeostasis of cancer cells for survival and growth. Intriguingly, this phenomenon provides a basis for synthetic lethal targeted therapies simultaneously administered to standard radiochemotherapy. In this review, we summarize current knowledge about the cell adhesion resistome and highlight targeting strategies to override CAM-RR and CAM-DR.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of additive particles on mechanical, thermal, and cell functioning properties of poly(methyl methacrylate) cement

PubMed Central

Khandaker, Morshed; Vaughan, Melville B; Morris, Tracy L; White, Jeremiah J; Meng, Zhaotong

2014-01-01

The most common bone cement material used clinically today for orthopedic surgery is poly(methyl methacrylate) (PMMA). Conventional PMMA bone cement has several mechanical, thermal, and biological disadvantages. To overcome these problems, researchers have investigated combinations of PMMA bone cement and several bioactive particles (micrometers to nanometers in size), such as magnesium oxide, hydroxyapatite, chitosan, barium sulfate, and silica. A study comparing the effect of these individual additives on the mechanical, thermal, and cell functional properties of PMMA would be important to enable selection of suitable additives and design improved PMMA cement for orthopedic applications. Therefore, the goal of this study was to determine the effect of inclusion of magnesium oxide, hydroxyapatite, chitosan, barium sulfate, and silica additives in PMMA on the mechanical, thermal, and cell functional performance of PMMA. American Society for Testing and Materials standard three-point bend flexural and fracture tests were conducted to determine the flexural strength, flexural modulus, and fracture toughness of the different PMMA samples. A custom-made temperature measurement system was used to determine maximum curing temperature and the time needed for each PMMA sample to reach its maximum curing temperature. Osteoblast adhesion and proliferation experiments were performed to determine cell viability using the different PMMA cements. We found that flexural strength and fracture toughness were significantly greater for PMMA specimens that incorporated silica than for the other specimens. All additives prolonged the time taken to reach maximum curing temperature and significantly improved cell adhesion of the PMMA samples. The results of this study could be useful for improving the union of implant-PMMA or bone-PMMA interfaces by incorporating nanoparticles into PMMA cement for orthopedic and orthodontic applications. PMID:24920906

Effect of additive particles on mechanical, thermal, and cell functioning properties of poly(methyl methacrylate) cement.

PubMed

Khandaker, Morshed; Vaughan, Melville B; Morris, Tracy L; White, Jeremiah J; Meng, Zhaotong

2014-01-01

The most common bone cement material used clinically today for orthopedic surgery is poly(methyl methacrylate) (PMMA). Conventional PMMA bone cement has several mechanical, thermal, and biological disadvantages. To overcome these problems, researchers have investigated combinations of PMMA bone cement and several bioactive particles (micrometers to nanometers in size), such as magnesium oxide, hydroxyapatite, chitosan, barium sulfate, and silica. A study comparing the effect of these individual additives on the mechanical, thermal, and cell functional properties of PMMA would be important to enable selection of suitable additives and design improved PMMA cement for orthopedic applications. Therefore, the goal of this study was to determine the effect of inclusion of magnesium oxide, hydroxyapatite, chitosan, barium sulfate, and silica additives in PMMA on the mechanical, thermal, and cell functional performance of PMMA. American Society for Testing and Materials standard three-point bend flexural and fracture tests were conducted to determine the flexural strength, flexural modulus, and fracture toughness of the different PMMA samples. A custom-made temperature measurement system was used to determine maximum curing temperature and the time needed for each PMMA sample to reach its maximum curing temperature. Osteoblast adhesion and proliferation experiments were performed to determine cell viability using the different PMMA cements. We found that flexural strength and fracture toughness were significantly greater for PMMA specimens that incorporated silica than for the other specimens. All additives prolonged the time taken to reach maximum curing temperature and significantly improved cell adhesion of the PMMA samples. The results of this study could be useful for improving the union of implant-PMMA or bone-PMMA interfaces by incorporating nanoparticles into PMMA cement for orthopedic and orthodontic applications.

Effects of Antioxidant Components of AREDS Vitamins and Zinc Ions on Endothelial Cell Activation: Implications for Macular Degeneration

PubMed Central

Zeng, Shemin; Hernández, Jasmine

2012-01-01

Purpose. To investigate whether the benefit of Age-Related Eye Disease Study (AREDS) formula multivitamins and zinc in the progression of age-related macular degeneration (AMD) may occur through inhibiting inflammatory events in the choroid. Methods. Mouse C166 endothelial cells (ECs) and, for some experiments, human retinal pigment epithelium (RPE)–choroid organ cultures were treated with AREDS multivitamin solution (MVS) or ZnCl2. The cytotoxicity of MVS was evaluated using a lactate dehydrogenase colorimetric assay. Cell motility was assessed using a scratch assay. Macrophage adhesion to EC monolayers or ICAM-1 protein was determined after MVS and zinc treatment and with or without lipopolysaccharide (LPS). Quantitative reverse transcription PCR and Western blot analysis were used to determine the effects of MVS on the expression of proinflammatory molecules in treated and untreated cells. Results. AREDS MVS and zinc did not affect C166 EC viability until the 56th hour after treatment. Scratch assays showed partial inhibition of MVS and zinc on EC migration. In cell adhesion assays, MVS and zinc decreased the number of macrophages bound to EC and to ICAM-1 protein. Quantitative PCR showed that LPS increased the expression of ICAM-1 in both C166 and human RPE-choroid cultures, which was partially offset by MVS and zinc. MVS and zinc also mitigated LPS-induced ICAM-1 protein expression on Western blot analysis. Conclusions. Treatment with AREDS MVS and zinc may affect both angiogenesis and endothelial-macrophage interactions. These results suggest that AREDS vitamins and zinc ions may slow the progression of AMD, in part through the attenuation of EC activation. PMID:22247465

Effects of antioxidant components of AREDS vitamins and zinc ions on endothelial cell activation: implications for macular degeneration.

PubMed

Zeng, Shemin; Hernández, Jasmine; Mullins, Robert F

2012-02-01

To investigate whether the benefit of Age-Related Eye Disease Study (AREDS) formula multivitamins and zinc in the progression of age-related macular degeneration (AMD) may occur through inhibiting inflammatory events in the choroid. Mouse C166 endothelial cells (ECs) and, for some experiments, human retinal pigment epithelium (RPE)-choroid organ cultures were treated with AREDS multivitamin solution (MVS) or ZnCl(2). The cytotoxicity of MVS was evaluated using a lactate dehydrogenase colorimetric assay. Cell motility was assessed using a scratch assay. Macrophage adhesion to EC monolayers or ICAM-1 protein was determined after MVS and zinc treatment and with or without lipopolysaccharide (LPS). Quantitative reverse transcription PCR and Western blot analysis were used to determine the effects of MVS on the expression of proinflammatory molecules in treated and untreated cells. AREDS MVS and zinc did not affect C166 EC viability until the 56th hour after treatment. Scratch assays showed partial inhibition of MVS and zinc on EC migration. In cell adhesion assays, MVS and zinc decreased the number of macrophages bound to EC and to ICAM-1 protein. Quantitative PCR showed that LPS increased the expression of ICAM-1 in both C166 and human RPE-choroid cultures, which was partially offset by MVS and zinc. MVS and zinc also mitigated LPS-induced ICAM-1 protein expression on Western blot analysis. Treatment with AREDS MVS and zinc may affect both angiogenesis and endothelial-macrophage interactions. These results suggest that AREDS vitamins and zinc ions may slow the progression of AMD, in part through the attenuation of EC activation.

Optimal conjugation of catechol group onto hyaluronic acid in coronary stent substrate coating for the prevention of restenosis.

PubMed

Lih, Eugene; Choi, Seul Gi; Ahn, Dong June; Joung, Yoon Ki; Han, Dong Keun

2016-01-01

Although endovascular stenting has been used as an interventional therapy to treat cardio- and cerebro-vascular diseases, it is associated with recurrent vascular diseases following stent thrombosis and in-stent restenosis. In this study, a metallic stent was coated with dopamine-conjugated hyaluronic acid with different ratios of catechol group to improve hemocompatibility and re-endothelialization. Especially, we were interested in how much amount of catechol group is appropriate for the above-mentioned purposes. Therefore, a series of dopamine-conjugated hyaluronic acid conjugates with different ratios of catechol group were synthesized via a carbodiimide coupling reaction. Dopamine-conjugated hyaluronic acid conjugates were characterized with 1 H-nuclear magnetic resonance and Fourier transform infrared spectroscopy, and the amount of catechol group in dopamine-conjugated hyaluronic acid was measured by ultraviolet spectrometer. Co-Cr substrates were polished and coated with various dopamine-conjugated hyaluronic acid conjugates under pH 8.5. Dopamine-conjugated hyaluronic acid amounts on the substrate were quantified by micro-bicinchoninic acid assay. Surface characteristics of dopamine-conjugated hyaluronic-acid-coated Co-Cr were evaluated by water contact angle, scanning electron microscopy, and atomic force microscopy. The hemocompatibility of the surface-modified substrates was assessed by protein adsorption and platelet adhesion tests. Adhesion and activation of platelets were confirmed with scanning electron microscopy and lactate dehydrogenase assay. Human umbilical vein endothelial cells were cultured on the substrates, and the viability, adhesion, and proliferation were investigated through cell counting kit-8 assay and fluorescent images. Obtained results demonstrated that optimal amounts of catechol group (100 µmol) in the dopamine-conjugated hyaluronic acid existed in terms of various properties such as hemocompatibility and cellular responses.

Fibrin-Genipin Adhesive Hydrogel for Annulus Fibrosus Repair: Performance Evaluation with Large Animal Organ Culture, In Situ Biomechanics, and In Vivo Degradation Tests

PubMed Central

Likhitpanichkul, M.; Dreischarf, M.; Illien-Junger, S.; Walter, B. A.; Nukaga, T.; Long, R. G; Sakai, D.; Hecht, A. C.; Iatridis, J. C.

2015-01-01

Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation. PMID:25036053

Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests.

PubMed

Likhitpanichkul, M; Dreischarf, M; Illien-Junger, S; Walter, B A; Nukaga, T; Long, R G; Sakai, D; Hecht, A C; Iatridis, J C

2014-07-18

Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation.

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID:11772392

Protopine inhibits heterotypic cell adhesion in MDA-MB-231 cells through down-regulation of multi-adhesive factors.

PubMed

He, Kai; Gao, Jian-Li

2014-01-01

A Chinese herb Corydalis yanhusuo W.T. Wang that showed anticancer and anti-angiogenesis effects in our previous studies was presented for further studies. In the present study, we studied the anticancer proliferation and adhesion effects of five alkaloids which were isolated from Corydalis yanhusuo. MTT dose response curves, cell migration assay, cell invasion assay, as well as three types of cell adhesive assay were performed on MDA-MB-231 human breast cancer cells. The mechanism of the compounds on inhibiting heterotypic cell adhesion were further explored by determining the expression of epidermal growth factor receptor (EGFR), Intercellular adhesion molecule 1 (ICAM-1), αv-integrin, β1-integrin and β5-integrin by western blotting assay. In five tested alkaloids, only protopine exhibited anti-adhesive and anti-invasion effects in MDA-MB-231 cells, which contributed to the anti-metastasis effect of Corydalis yanhusuo. The results showed that after treatment with protopine for 90 min, the expression of EGFR, ICAM-1, αv-integrin, β1-integrin and β5-integrin were remarkably reduced. The present results suggest that protopine seems to inhibit the heterotypic cell adhesion between MDA-MB-231 cells, and human umbilical vein endothelial cells by changing the expression of adhesive factors.

Saccharomyces boulardii expresses neuraminidase activity selective for α2,3-linked sialic acid that decreases Helicobacter pylori adhesion to host cells.

PubMed

Sakarya, Serhan; Gunay, Necati

2014-10-01

Helicobacter pylori is a major causative agent of gastritis and peptic ulcer disease and is an established risk factor for gastric malignancy. Antibiotic combination therapy can eradicate H. pylori. As these same regimens can evoke adverse effects and resistance, new alternative therapies or adjunctive treatments are needed. A probiotic approach may provide a novel strategy for H. pylori treatment. In the current study, two probiotic bacteria, Lactobacillus acidophilus and Lactobacillus reuteri, and a probiotic yeast, Saccharomyces boulardii, were evaluated for their ability to influence H. pylori viability, adherence to gastric and duodenal cells, as well as the effect of S. boulardii on cell surface expression of sialic acid. Our results indicate that S. boulardii contains neuraminidase activity selective for α(2-3)-linked sialic acid. This neuraminidase activity removes surface α(2-3)-linked sialic acid, the ligand for the sialic acid-binding H. pylori adhesin, which in turn, inhibits H. pylori adherence to duodenal epithelial cells. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Assessment and comparison of probiotic potential of four Lactobacillus species isolated from feces samples of Iranian infants.

PubMed

Halimi, Shahnaz; Mirsalehian, Akbar

2016-02-01

The probiotic potential of Lactobacillus species isolated from infant feces was investigated. For this study, the antibiotic susceptibility, tolerance in gut-related conditions, antimicrobial activity, and ability to adhere to a human colorectal adenocarcinoma cell line (Caco-2 cells) of four common Lactobacillus species (Lactobacillus paracasei [n = 15], Lactobacillus rhamnosus [n = 45], Lactobacillus gasseri [n = 20] and Lactobacillus fermentum [n = 18]) were assessed. Most isolates that which were sensitive to imipenem, ampicillin, gentamycin, erythromycin and tetracycline were selected for other tests. L. gasseri isolates had the greatest sensitivity to gastric and intestinal fluids (<10% viability). L. fermentum (FH5, FH13 and FH18) had the highest adhesion to Caco-2 cells. The lowest antibacterial activity against pathogenic bacteria was shown by L. gasseri strains in spot tests. Furthermore, non-adjusted cell-free culture supernatants with low pH had greater antimicrobial activity, which was related to organic acid. The results showed that some isolates of L. rhamnosus and L. fermentum are suitable for use as a probiotic. © 2015 The Societies and John Wiley & Sons Australia, Ltd.

Bromelain Reversibly Inhibits Invasive Properties of Glioma Cells

PubMed Central

Tysnes, Berit B; Maurer, H Rainer; Porwol, Torsten; Probst, Beatrice; Bjerkvig, Rolf; Hoover, Frank

2001-01-01

Abstract Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that α3 and β1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a trans-activating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation. PMID:11774029

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth.

PubMed

Rao, Qing; Wang, Ji-Ying; Meng, Jihong; Tang, Kejing; Wang, Yanzhong; Wang, Min; Xing, Haiyan; Tian, Zheng; Wang, Jianxiang

2011-09-01

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.

Application of High-Impact Polystyrene (HIPS) as a Graphene Nanoparticle Reinforced Composite Thermoplastic Adhesive

NASA Astrophysics Data System (ADS)

Stitt, Erik

Adhesive bonding is a more efficient joining method for composites than traditional mechanical fasteners and provides advantages in weight reduction, simplicity, and cost. In addition, the utilization of mechanical fasteners introduces stress concentrations and damage to the fiber-matrix interface. Adhesive bonding with thermoset polymers distributes mechanical loads but also makes disassembly for repair and recycling difficult. The ability to utilize thermoplastic polymers as adhesives offers an approach to address these limitations and can even produce a reversible adhesive joining technology through combining conductive nanoparticles with a thermoplastic polymer. The incorporation of the conductive nanoparticles allows for selective heating of the adhesive via exposure to electromagnetic (EM) radiation and simultaneously can augment the mechanical properties of the adhesive and the adhesive joint. This approach provides a versatile mechanism for efficiently creating and reversing structural adhesive joints across a wide range of materials. In this work, a high-impact polystyrene (HIPS) co-polymer containing butadiene as a toughness modifier is compounded with graphene nano-platelets (GnP) for investigation as a thermoplastic adhesive. The properties of the bulk composite adhesive are tailored by altering the morphology, dispersion, and concentration of GnP. The thermal response of the material to EM radiation in the microwave frequency spectrum was investigated and optimized. Surface treatments of the adhesive films were explored to enhance the viability of this nanoparticle thermoplastic polymer to function as a reversible adhesive. As a result, it has been shown that lap-shear strengths of multi-material joints produced from aforementioned thermoplastic adhesives were comparable to similar thermoset bonded joints.

Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

PubMed

Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

2015-01-01

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

PubMed

Kaminski, Alexander; Ma, Nan; Donndorf, Peter; Lindenblatt, Nicole; Feldmeier, Gregor; Ong, Lee-Lee; Furlani, Dario; Skrabal, Christian A; Liebold, Andreas; Vollmar, Brigitte; Steinhoff, Gustav

2008-01-01

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

PubMed

Sergé, Arnauld

2016-01-01

The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.

The effect of ultrasound-related stimuli on cell viability in microfluidic channels

PubMed Central

2013-01-01

Background In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Results Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0–29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. Conclusion The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells. PMID:23809777

The effect of ultrasound-related stimuli on cell viability in microfluidic channels.

PubMed

Ankrett, Dyan N; Carugo, Dario; Lei, Junjun; Glynne-Jones, Peter; Townsend, Paul A; Zhang, Xunli; Hill, Martyn

2013-06-28

In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0-29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.

Cellular injuries of spray-dried Lactobacillus spp. isolated from kefir and their impact on probiotic properties.

PubMed

Golowczyc, Marina A; Silva, Joana; Teixeira, Paula; De Antoni, Graciela L; Abraham, Analía G

2011-01-05

The injuries caused by spray drying (SD) of three potential probiotic lactobacilli isolated from kefir grains and the impact on some probiotic properties, were evaluated. Results demonstrated that Lactobacillus plantarum 83114 and L. kefir 8321 showed a slight reduction of viability (0.11 and 0.29 log CFU/ml respectively) after SD process, and L. kefir 8348 was found to be more sensitive to the process with a reduction in viability of 0.70 log CFU/ml. Neither membrane damage, evaluated by increased sensitivity to NaCl, lysozyme, bile salt and penicillin G, nor changes in acidifying activity in MRS and milk by lactobacilli were detected after SD. L. plantarum 83114 and L. kefir 8321 after SD did not lose their capacity to adhere to intestinal cells. Nevertheless, L. kefir 8348 showed a significant loss of adhesion capacity after SD. In addition, rehydrated spray-dried L. kefir 8321 retained the ability to protect against Salmonella invasion of intestinal cells. This effect was observed when L. kefir is co-incubated with Salmonella before invasion assay. This work shows that the membrane integrity evaluated by indirect methods and some probiotic properties of lactobacilli isolated from kefir did not change significantly after SD, and these powders could be used in functional foods applications. Copyright © 2010 Elsevier B.V. All rights reserved.

The Interplay between Entamoeba and Enteropathogenic Bacteria Modulates Epithelial Cell Damage

PubMed Central

Galván-Moroyoqui, José Manuel; Domínguez-Robles, M. del Carmen; Franco, Elizabeth; Meza, Isaura

2008-01-01

Background Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined. Methodology Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified. Principal Findings E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica. Conclusions Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage. Significance The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon. PMID:18648517

Bio-Orthogonally Crosslinked, Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation.

PubMed

Madl, Christopher M; Katz, Lily M; Heilshorn, Sarah C

2016-06-07

Covalently-crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross-react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio-orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin-like proteins (ELPs) are developed that cross-link via strain-promoted azide-alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC-crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC-ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin-derived arginine-glycine-aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.

BMS-777607 promotes megakaryocytic differentiation and induces polyploidization in the CHRF-288-11 cells.

PubMed

Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Taya, Masahito

2015-04-01

Introduction of a polyploidy inducer is a promising strategy to achieve a high level of polyploidization during megakaryocytic (MK) differentiation. Here, we report that a multi-kinase inhibitor, BMS-777607, is a potent polyploidy inducer for elevating high ploidy cell formation in the MK-differentiated CHRF-288-11 (CHRF) cells. Our result showed that BMS-777607 strongly inhibited cell division without affecting cell viability when detected at day 1 after treatment. As a consequence, the high ploidy (≥8N) cells were accumulated in culture for 8 days, with an increase from 16.2 to 75.2 % of the total cell population. The elevated polyploidization was accompanied by the increased expression level of MK marker, CD41 (platelet glycoprotein IIb/IIIa, GPIIb/IIIa), suggesting that BMS-777607 promoted both polyploidization and commitment of MK-differentiated CHRF cells. Platelet-like fragments (PFs) were released by mature CHRF cells. Based on a flow cytometry assay, it was found that the PFs produced from BMS-777607-treated cells tended to have larger size and higher expression of GPIIb/IIIa, a receptor for platelet adhesion. Taken together, these results suggested that BMS-777607 promoted MK differentiation of CHRF cells and increased the functional property of platelet-like fragments.

Embryonic stem cells growing in 3-dimensions shift from reliance on the substrate to each other for mechanical support.

PubMed

Teo, Ailing; Lim, Mayasari; Weihs, Daphne

2015-07-16

Embryonic stem cells (ESCs) grow into three-dimensional (3D) spheroid structures en-route to tissue growth. In vitro spheroids can be controllably induced on a two-dimensional (2D) substrate with high viability. Here we use a method for inducing pluripotent embryoid body (EB) formation on flat polyacrylamide gels while simultaneously evaluating the dynamic changes in the mechano-biology of the growing 3D spheroids. During colony growth in 3D, pluripotency is conserved while the spheroid-substrate interactions change significantly. We correlate colony-size, cell-applied traction-forces, and expressions of cell-surface molecules indicating cell-cell and cell-substrate interactions, while verifying pluripotency. We show that as the colony size increases with time, the stresses applied by the spheroid to the gel decrease in the 3D growing EBs; control cells growing in 2D-monolayers maintain unvarying forces. Concurrently, focal-adhesion mediated cell-substrate interactions give way to E-cadherin cell-cell connections, while pluripotency. The mechano-biological changes occurring in the growing embryoid body are required for stabilization of the growing pluripotent 3D-structure, and can affect its potential uses including differentiation. This could enable development of more effective expansion, differentiation, and separation approaches for clinical purposes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nanochips of Tantalum Oxide Nanodots as artificial-microenvironments for monitoring Ovarian cancer progressiveness

NASA Astrophysics Data System (ADS)

Dhawan, Udesh; Wang, Ssu-Meng; Chu, Ying Hao; Huang, Guewha S.; Lin, Yan Ren; Hung, Yao Ching; Chen, Wen Liang

2016-08-01

Nanotopography modulates cell characteristics and cell behavior. Nanotopological cues can be exploited to investigate the in-vivo modulation of cell characteristics by the cellular microenvironment. However, the studies explaining the modulation of tumor cell characteristics and identifying the transition step in cancer progressiveness are scarce. Here, we engineered nanochips comprising of Tantalum oxide nanodot arrays of 10, 50, 100 and 200 nm as artificial microenvironments to study the modulation of cancer cell behavior. Clinical samples of different types of Ovarian cancer at different stages were obtained, primary cultures were established and then seeded on different nanochips. Immunofluorescence (IF) was performed to compare the morphologies and cell characteristics. Indices corresponding to cell characteristics were defined. A statistical comparison of the cell characteristics in response to the nanochips was performed. The cells displayed differential growth parameters. Morphology, Viability, focal adhesions, microfilament bundles and cell area were modulated by the nanochips which can be used as a measure to study the cancer progressiveness. The ease of fabrication of nanochips ensures mass-production. The ability of the nanochips to act as artificial microenvironments and modulate cell behavior may lead to further prospects in the markerless monitoring of the progressiveness and ultimately, improving the prognosis of Ovarian cancer.

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin*

PubMed Central

Kwak, Tae Kyoung; Lee, Mi-Sook; Ryu, Jihye; Choi, Yoon-Ju; Kang, Minkyung; Jeong, Doyoung; Lee, Jung Weon

2012-01-01

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration. PMID:22761432

A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

PubMed

Miyoshi, Yukihiro; Okada, Sanae; Uchimura, Tai; Satoh, Eiichi

2006-07-01

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

Synthesis and characterization of chitosan-alginate scaffolds for seeding human umbilical cord derived mesenchymal stem cells.

PubMed

Kumbhar, Sneha G; Pawar, S H

2016-01-01

Chitosan and alginate are two natural and accessible polymers that are known to be biocompatible, biodegradable and possesses good antimicrobial activity. When combined, they exhibit desirable characteristics and can be created into a scaffold for cell culture. In this study interaction of chitosan-alginate scaffolds with mesenchymal stem cells are studied. Mesenchymal stem cells were derived from human umbilical cord tissues, characterized by flow cytometry and other growth parameters studied as well. Proliferation and viability of cultured cells were studied by MTT Assay and Trypan Blue dye exclusion assay. Besides chitosan-alginate scaffold was prepared by freeze-drying method and characterized by FTIR, SEM and Rheological properties. The obtained 3D porous structure allowed very efficient seeding of hUMSCs that are able to inhabit the whole volume of the scaffold, showing good adhesion and proliferation. These materials showed desirable rheological properties for facile injection as tissue scaffolds. The results of this study demonstrated that chitosan-alginate scaffold may be promising biomaterial in the field of tissue engineering, which is currently under a great deal of examination for the development and/or restoration of tissue and organs. It combines the stem cell therapy and biomaterials.

A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency

PubMed Central

Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

2015-01-01

Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose–asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes. PMID:25848252

Novel electrospun nanofibers of modified gelatin-tyrosine in cartilage tissue engineering.

PubMed

Agheb, Maria; Dinari, Mohammad; Rafienia, Mohammad; Salehi, Hossein

2017-02-01

In natural cartilage tissues, chondrocytes are linked to extracellular matrix (ECM) through cell-surface binding proteins. Surface modification of gelatin can provide a new generation of biopolymers and fibrous scaffolds with chemical, mechanical, and biological properties. In this study tyrosine protein and 1,2,3-triazole ring were utilized to functionalize gelatin without Cu catalyst. Their molecular structure was characterized by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy ( 1 HNMR). Chemical cross-linkers such as glutaraldehyde (GA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) were used to electrospin the modified gelatin. The modification of gelatin and cross-linking effects were confirmed by scanning electron microscopy (SEM), contact angle measurement, and mechanical tests. MTT assay using chondrocyte cells showed cell viability of electrospun modified gelatin scaffolds. In vitro cell culture studies showed that electrospun engineered protein scaffolds would support the attachment and growth of cells. The results also showed that cross-linked nanofibers with EDC/NHS could be considered excellent matrices in cell adhesion and proliferation before electrospinning process and their potential substrate in tissue engineering applications, especially in the field of cartilage engineering. Copyright © 2016. Published by Elsevier B.V.

Synergistic effect of two cell recognition systems: glycosphingolipid-glycosphingolipid interaction and integrin receptor interaction with pericellular matrix protein.

PubMed

Kojima, N; Hakomori, S

1991-12-01

GM3-expressing cells adhere, spread and migrate on plastic plates coated with Gg3, LacCer and Gb4, but not with other glycosphingolipids (GSLs). Thus, cell adhesion, spreading and migration through GSL-GSL interaction occur in an analogous fashion to the interaction of cells with adhesive matrix proteins [AP, e.g. fibronectin (FN), laminin (LN)] through their integrin receptors. In this study, the adhesion of two GM3-expressing cell lines (B16 melanoma and HEL299 fibroblast) on plastic plates co-coated with GSL plus AP is compared with adhesion on plates coated with GSL (Gg3 or LacCer) alone, or coated with AP alone. Results show that: (i) cell adhesion on GSL-coated plates takes place earlier in the incubation period than that on AP-coated plates; (ii) cell adhesion, as well as spreading, was greatly enhanced (in terms of strength and rapidity) on plates co-coated with GSL plus AP; (iii) repulsion (negative adhesion) of cells was observed on plates co-coated with AP plus N-acetyl-GM3 (NAcGM3) and was presumably based on repulsive NAcGM3-NAcGM3 interaction; (iv) GM3-dependent cell adhesion on GSL-coated plates, as well as synergistic promotion of cell adhesion (based on the GSL-GSL and AP-integrin systems), was suppressed by incubation of cells with anti-GM3 monoclonal antibody DH2 or sialidase. Synergistic adhesion of cells on GSL/AP co-coated plates was less inhibited by incubation with peptide sequences RGDS or YIGSR than was adhesion on plates coated with AP alone.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo studies of sickle red blood cells.

PubMed

Kaul, Dhananjay K; Fabry, Mary E

2004-03-01

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

PubMed Central

Arregui, Carlos O.; Balsamo, Janne; Lilien, Jack

1998-01-01

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions. PMID:9813103

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings.

PubMed

Kovacs, Boglarka; Patko, Daniel; Szekacs, Inna; Orgovan, Norbert; Kurunczi, Sandor; Sulyok, Attila; Khanh, Nguyen Quoc; Toth, Balazs; Vonderviszt, Ferenc; Horvath, Robert

2016-09-15

Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. In the present work, we show for the first time that. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1.

PubMed

Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

2014-06-01

T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130,000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1-calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.

Redistribution of Adhesive Forces through Src/FAK Drives Contact Inhibition of Locomotion in Neural Crest.

PubMed

Roycroft, Alice; Szabó, András; Bahm, Isabel; Daly, Liam; Charras, Guillaume; Parsons, Maddy; Mayor, Roberto

2018-06-04

Contact inhibition of locomotion is defined as the behavior of cells to cease migrating in their former direction after colliding with another cell. It has been implicated in multiple developmental processes and its absence has been linked to cancer invasion. Cellular forces are thought to govern this process; however, the exact role of traction through cell-matrix adhesions and tension through cell-cell adhesions during contact inhibition of locomotion remains unknown. Here we use neural crest cells to address this and show that cell-matrix adhesions are rapidly disassembled at the contact between two cells upon collision. This disassembly is dependent upon the formation of N-cadherin-based cell-cell adhesions and driven by Src and FAK activity. We demonstrate that the loss of cell-matrix adhesions near the contact leads to a buildup of tension across the cell-cell contact, a step that is essential to drive cell-cell separation after collision. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Tuning cell adhesive properties via layer-by-layer assembly of chitosan and alginate

PubMed Central

Silva, Joana M.; García, José R.; Reis, Rui L.; García, Andrés J.; Mano, João F.

2017-01-01

Understanding the mechanisms controlling cell-multilayer film interactions is crucial to the successful engineering of these coatings for biotechnological and biomedical applications. Herein, we present a strategy to tune the cell adhesive properties of multilayers based on marine polysaccharides with and without cross-linking and/or coating with extracellular matrix proteins. Chemical cross-linking of multilayers improved mechanical properties of the coatings but also elicited changes in surface chemistry that alter the adhesion of human umbilical vein endothelial cells. We evaluated a strategy to decouple the mechanical and chemical properties of these films, enabling the transition from cell-adhesive to cell-resistant multilayers. Addition of chitosan/alginate multilayers on top of cross-linked films decreased endothelial cell adhesion, spreading, and proliferation to similar levels as uncross-linked films. Our findings highlight the key role of surface chemistry in cell-multilayer film interactions, and these engineered nanocoatings represent a tunable model of cell adhesive and non-adhesive multilayered films. PMID:28126597

Molecular analysis of antigen-independent adhesion forces between T and B lymphocytes.

PubMed Central

Amblard, F; Auffray, C; Sekaly, R; Fischer, A

1994-01-01

The low-affinity interactions underlying antigen recognition by T-cell receptors (TCRs) are thought to involve antigen-independent adhesion mechanisms. Using a hydrodynamic approach, we found that antigen-independent adhesion occurred between human B cells and resting T cells in a transient and temperature-dependent fashion. The mean cell-cell adhesion force was 0.32 x 10(-9) N and was generated by similar contributions (0.16 x 10(-9) N) of the LFA-1- and CD2-dependent adhesion pathways. After T-cell stimulation with a phorbol ester, the force contributed by LFA-1 was drastically increased, while that of CD2 was unaffected. We propose that weak receptor-mediated adhesion initiates antigen-independent intercellular contacts required for antigen recognition by the TCR and is upregulated following TCR engagement. The method used permits adhesion forces between living cells to be resolved at the molecular level and should prove valuable for the rapid assessment of interaction forces between various types of cells and cell-sized particles. Images PMID:7909604

Cell-cell adhesion in the cnidaria: insights into the evolution of tissue morphogenesis.

PubMed

Magie, Craig R; Martindale, Mark Q

2008-06-01

Cell adhesion is a major aspect of cell biology and one of the fundamental processes involved in the development of a multicellular animal. Adhesive mechanisms, both cell-cell and between cell and extracellular matrix, are intimately involved in assembling cells into the three-dimensional structures of tissues and organs. The modulation of adhesive complexes could therefore be seen as a central component in the molecular control of morphogenesis, translating information encoded within the genome into organismal form. The availability of whole genomes from early-branching metazoa such as cnidarians is providing important insights into the evolution of adhesive processes by allowing for the easy identification of the genes involved in adhesion in these organisms. Discovery of the molecular nature of cell adhesion in the early-branching groups, coupled with comparisons across the metazoa, is revealing the ways evolution has tinkered with this vital cellular process in the generation of the myriad forms seen across the animal kingdom.

Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

NASA Astrophysics Data System (ADS)

Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

1991-08-01

The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts

PubMed Central

Apostolopoulou, Maria; Ligon, Lee

2012-01-01

In the early stages of breast cancer metastasis, epithelial cells penetrate the basement membrane and invade the surrounding stroma, where they encounter fibroblasts. Paracrine signaling between fibroblasts and epithelial tumor cells contributes to the metastatic cascade, but little is known about the role of adhesive contacts between these two cell types in metastasis. Here we show that MCF-7 breast cancer epithelial cells and normal breast fibroblasts form heterotypic adhesions when grown together in co-culture, as evidenced by adhesion assays. PCR and immunoblotting show that both cell types express multiple members of the cadherin superfamily, including the atypical cadherin, cadherin-23, when grown in isolation and in co-culture. Immunocytochemistry experiments show that cadherin-23 localizes to homotypic adhesions between MCF-7 cells and also to heterotypic adhesions between the epithelial cells and fibroblasts, and antibody inhibition and RNAi experiments show that cadherin-23 plays a role in mediating these adhesive interactions. Finally, we show that cadherin-23 is upregulated in breast cancer tissue samples, and we hypothesize that heterotypic adhesions mediated by this atypical cadherin may play a role in the early stages of metastasis. PMID:22413011

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

PubMed

Chen, Jiahuan; Ganguly, Anutosh; Mucsi, Ashley D; Meng, Junchen; Yan, Jiacong; Detampel, Pascal; Munro, Fay; Zhang, Zongde; Wu, Mei; Hari, Aswin; Stenner, Melanie D; Zheng, Wencheng; Kubes, Paul; Xia, Tie; Amrein, Matthias W; Qi, Hai; Shi, Yan

2017-02-01

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner. © 2017 Chen et al.

Glycolytic inhibitors 2-deoxyglucose and 3-bromopyruvate synergize with photodynamic therapy respectively to inhibit cell migration.

PubMed

Feng, Xiaolan; Wang, Pan; Liu, Quanhong; Zhang, Ting; Mai, Bingjie; Wang, Xiaobing

2015-06-01

Most cancer cells have the specially increased glycolytic phenotype, which makes this pathway become an attractive therapeutic target. Although glycolytic inhibitor 2-deoxyglucose (2-DG) has been demonstrated to potentiate the cytotoxicity of photodynamic therapy (PDT), the impacts on cell migration after the combined treatment has never been reported yet. The present study aimed to analyze the influence of glycolytic inhibitors 2-DG and 3-bromopyruvate (3-BP) combined with Ce6-PDT on cell motility of Triple Negative Breast Cancer MDA-MB-231 cells. As determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium-bromide-Tetraz-olium (MTT) assay, more decreased cell viability was observed in 2-DG + PDT and 3-BP + PDT groups when compared with either monotherapy. Under optimal conditions, synergistic potentiation on cell membrane destruction and the decline of cell adhesion and cells migratory ability were observed in both 2-DG + PDT and 3-BP + PDT by electron microscope observation (SEM), wound healing and trans-well assays. Besides, serious microfilament network collapses as well as impairment of matrix metalloproteinases-9 (MMP-9) were notably improved after the combined treatments by immunofluorescent staining. These results suggest that 2-DG and 3-BP can both significantly potentiated Ce6-PDT efficacy of cell migration inhibition.

CD147-targeting siRNA inhibits cell-matrix adhesion of human malignant melanoma cells by phosphorylating focal adhesion kinase.

PubMed

Nishibaba, Rie; Higashi, Yuko; Su, Juan; Furukawa, Tatsuhiko; Kawai, Kazuhiro; Kanekura, Takuro

2012-01-01

CD147/basigin, highly expressed on the surface of malignant tumor cells including malignant melanoma (MM) cells, plays a critical role in the invasiveness and metastasis of MM. Metastasis is an orchestrated process comprised of multiple steps including adhesion and invasion. Integrin, a major adhesion molecule, co-localizes with CD147/basigin on the cell surface. Using the human MM cell line A375 that highly expresses CD147/basigin, we investigated whether CD147/basigin is involved in adhesion in association with integrin. CD147/basigin was knocked-down using siRNA targeting CD147 to elucidate the role of CD147/basigin. Cell adhesion was evaluated by adhesion assay on matrix-coated plates. The localization of integrin was inspected under a confocal microscope and the expression and phosphorylation of focal adhesion kinase (FAK), a downstream kinase of integrin, were examined by western blot analysis. Silencing of CD147/basigin in A375 cells by siRNA induced the phosphorylation of FAK at Y397. Integrin identified on the surface of parental cells was distributed in a speckled fashion in the cytoplasm of CD147 knockdown cells, resulting in morphological changes from a round to a polygonal shape with pseudopodial protrusions. Silencing of CD147/basigin in A375 cells clearly weakened their adhesiveness to collagen I and IV. Our results suggest that CD147/basigin regulates the adhesion of MM cells to extracellular matrices and of integrin β1 signaling via the phosphorylation of FAK. © 2011 Japanese Dermatological Association.

Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma.

PubMed

Lyons, A J; Jones, J

2007-08-01

Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells to form metastases at distant sites. These processes require alterations in cell-cell and cell-extracellular matrix interactions. As cell adhesion molecules play a role in cell-cell and cell-extracellular matrix adhesion and interactions they are involved in the process of tumour invasion and metastases. In epithelial tissues, receptors of the integrin family mediate adhesion to the adjacent matrix whereas cadherins largely mediate intercellular adhesion. These and other cell adhesion molecules such as intercellular adhesion molecule-1, CD44, dystroglycans and selectins, are involved and undergo changes in carcinomas, which provide possible targets for anti-cancer drug treatments. In the extracellular matrix that is associated with tumours, laminin 5, oncofetal fibronectin and tenascin C appear. The degree of expression of some of these moieties indicates prognosis in oral cancer and offer targets for antibody-directed radiotherapy. Metalloproteases which degrade the extracellular matrix are increased in carcinomas, and their activity is necessary for tumour angiogenesis and consequent invasion and metastases. Metalloprotease inhibitors have begun to produce decreases in mortality in clinical trials. This report provides a brief overview of our current understanding of cell adhesion molecules, the extracellular matrix, tumour invasion and metastasis.

Application of Organosilane Monolayer Template to Quantitative Evaluation of Cancer Cell Adhesive Ability

NASA Astrophysics Data System (ADS)

Tanii, Takashi; Sasaki, Kosuke; Ichisawa, Kota; Demura, Takanori; Beppu, Yuichi; Vu, Hoan Anh; Thanh Chi, Hoan; Yamamoto, Hideaki; Sato, Yuko

2011-06-01

The adhesive ability of two human pancreatic cancer cell lines was evaluated using organosilane monolayer templates (OMTs). Using the OMT, the spreading area of adhered cells can be limited, and this enables us to focus on the initial attachment process of adhesion. Moreover, it becomes possible to arrange the cells in an array and to quantitatively evaluate the number of attached cells. The adhesive ability of the cancer cells cultured on the OMT was controlled by adding (-)-epigallocatechin-3-gallate (EGCG), which blocks a receptor that mediates cell adhesion and is overexpressed in cancer cells. Measurement of the relative ability of the cancer cells to attach to the OMT revealed that the ability for attachment decreased with increasing EGCG concentration. The results agreed well with the western blot analysis, indicating that the OMT can potentially be employed to evaluate the adhesive ability of various cancer cells.

Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

PubMed

Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

2016-07-07

The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

NCAM polysialylation during adherence transitions: live cell monitoring using an antibody-mimetic EGFP-endosialidase and the viability dye DRAQ7.

PubMed

Smith, Paul J; Furon, Emeline; Wiltshire, Marie; Chappell, Sally; Patterson, Laurence H; Shnyder, Steven D; Falconer, Robert A; Errington, Rachel J

2013-07-01

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell-surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell-cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies. Copyright © 2013 International Society for Advancement of Cytometry.

Bone-repair properties of biodegradable hydroxyapatite nano-rod superstructures

NASA Astrophysics Data System (ADS)

D'Elía, Noelia L.; Mathieu, Colleen; Hoemann, Caroline D.; Laiuppa, Juan A.; Santillán, Graciela E.; Messina, Paula V.

2015-11-01

Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants.Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants. Electronic supplementary information (ESI) available: Calculation of roughness parameters Rz, Rz,max, and Rz, prom. Nano-HAp powder degradation after immersion in phosphate buffer (pH = 7.4). Optical phase contrast microphotographs of MSC adhesion on nano-HAp and nano-HAp/Co I coatings at different concentrations. Laser scanning confocal microphotographs of MSCs' α-SMA expression spreading on large amounts of nano-HAp (MI) coatings. Immunofluorescence microphotograph analysis by image software. See DOI: 10.1039/c5nr04850h

Nano- and Micro-Patterned S-, H-, and X-PDMS for Cell-Based Applications: Comparison of Wettability, Roughness, and Cell-Derived Parameters

PubMed Central

Scharin-Mehlmann, Marina; Häring, Aaron; Rommel, Mathias; Dirnecker, Tobias; Friedrich, Oliver; Frey, Lothar; Gilbert, Daniel F.

2018-01-01

Polydimethylsiloxane (PDMS) is a promising biomaterial for generating artificial extracellular matrix (ECM) like patterned topographies, yet its hydrophobic nature limits its applicability to cell-based approaches. Although plasma treatment can enhance the wettability of PDMS, the surface is known to recover its hydrophobicity within a few hours after exposure to air. To investigate the capability of a novel PDMS-type (X-PDMS) for in vitro based assessment of physiological cell properties, we designed and fabricated plane as well as nano- and micrometer-scaled pillar-patterned growth substrates using the elastomer types S-, H- and X-PDMS, which were fabricated from commercially available components. Most importantly, we compared X-PDMS based growth substrates which have not yet been investigated in this context with H- as well as well-known S-PDMS based substrates. Due to its applicability to fabricating nanometer-sized topographic features with high accuracy and pattern fidelity, this material may be of high relevance for specific biomedical applications. To assess their applicability to cell-based approaches, we characterized the generated surfaces using water contact angle (WCA) measurement and atomic force microscopy (AFM) as indicators of wettability and roughness, respectively. We further assessed cell number, cell area and cellular elongation as indirect measures of cellular viability and adhesion by image cytometry and phenotypic profiling, respectively, using Calcein and Hoechst 33342 stained human foreskin fibroblasts as a model system. We show for the first time that different PDMS types are differently sensitive to plasma treatment. We further demonstrate that surface hydrophobicity changes along with changing height of the pillar-structures. Our data indicate that plane and structured X-PDMS shows cytocompatibility and adhesive properties comparable to the previously described elastomer types S- and H-PDMS. We conclude that nanometer-sized structuring of X-PDMS may serve as a powerful method for altering surface properties toward production of biomedical devices for cell-based applications. PMID:29765941

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

PubMed

Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

2015-03-02

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.

Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whelan, Jarrett T.; Wang, Lei; Chen, Jianming

2014-11-28

Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid Xmore » receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.« less