Mammalian Cell Adhesion Research Articles

Cell Compatible Trimethoprim-Decorated Iron Oxide Nanoparticles Bind Dihydrofolate Reductase for Magnetically Modulating Focal Adhesion of Mammalian Cells

On the basis of the high affinity binding of trimethoprim (TMP) to Escherichia coli dihydrofolate reductase (eDHFR), TMP-decorated iron oxide nanoparticles bind to eDHFR with high affinity and specificity, which allows magnetic modulation of focal adhesion of mammalian cells adhered to a surface. Besides being the first example of nanoparticles that selectively bind to eDHFR, the biocompatibility of the conjugate of TMP-iron oxide nanoparticles renders a convenient and versatile platform for investigating the cellular responses to specific, mechanical perturbation of proteins via a magnetic force.

Biosynthesis of Galactofuranose in Kinetoplastids: Novel Therapeutic Targets for Treating Leishmaniasis and Chagas' Disease

Cell surface proteins of parasites play a role in pathogenesis by modulating mammalian cell recognition and cell adhesion during infection. β-Galactofuranose (Galf) is an important component of glycoproteins and glycolipids found on the cell surface of Leishmania spp. and Trypanosoma cruzi. β-Galf-containing glycans have been shown to be important in parasite-cell interaction and protection against oxidative stress. Here, we discuss the role of β-Galf in pathogenesis and recent studies on the Galf-biosynthetic enzymes: UDP-galactose 4′ epimerase (GalE), UDP-galactopyranose mutase (UGM), and UDP-galactofuranosyl transferase (GalfT). The central role in Galf formation, its unique chemical mechanism, and the absence of a homologous enzyme in humans identify UGM as the most attractive drug target in the β-Galf-biosynthetic pathway in protozoan parasites.

Fabrication and Selective Functionalization of Amine-Reactive Polymer Multilayers on Topographically Patterned Microwell Cell Culture Arrays

We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. "Reactive" layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts.

Stereochemical effects of chiral monolayers on enhancing the resistance to mammalian cell adhesion

This work describes the different durations of surface confinement of adhered mammalian cells by monolayers comprised of enantiomers of bio-inert polyol-terminated alkanethiols. Enhanced resistance to protein adsorption and cell adhesion is obtained on monolayers formed by a racemic mixture of the enantiomeric alkanethiols.

Adhesamine, a new synthetic molecule, accelerates differentiation and prolongs survival of primary cultured mouse hippocampal neurons

Attachment to the substrate is essential for both survival and differentiation of various kinds of cells, such as neurons and epithelial cells. We recently found a small synthetic molecule, adhesamine, which boosts adhesion and growth of mammalian cells. In the present study, we applied adhesamine to primary cultured hippocampal neuronal cells and compared its effects with those of PLL (poly-L-lysine), which is widely used as a substrate for cell cultures. Neurons grown on adhesamine-coated coverslips survived for up to 1 month without a feeder layer of glial cells, and had greater viability than cells grown on PLL-coated coverslips. Morphological analysis revealed that neurons cultured with adhesamine exhibited earlier differentiation, i.e. earlier axonal outgrowth and dendritic maturation with enhanced neurite branching, than neurons cultured with PLL. Synaptic formation and postsynaptic responses were evident as early as 4 days in cells cultured with adhesamine. Acceleration of differentiation is mediated by earlier activation of the signalling pathways from heparan sulfate in the extracellular matrix to both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). Improved survival rates and accelerated maturation of neurons exposed to adhesamine suggest that this completely synthetic molecule may be a useful reagent for culturing neuronal cells.

Mechanical contributions to astrocyte adhesion using a novel in vitro model of catheter obstruction

Drainage and diversion of cerebrospinal fluid (CSF) through shunt systems is the most common treatment for hydrocephalus, but complications due to tissue obstruction of the catheter occur in up to 61% of patients. Although shunt systems have undergone limited technological advancements to resist mammalian cell adhesion, there is a need to further reduce adhesion that can exacerbate obstruction. The high intrinsic variability in clinical studies and an inability to predict chronic adhesion of host cells in vitro while maintaining the environmental conditions observed in hydrocephalus have impeded progress. We designed the hydrocephalus shunt catheter bioreactor (HSCB) to measure inflammatory cell adhesion under experimentally manipulated conditions of CSF pressure, pulsation rate, and flow rates. For a 20-h period, astrocytes were perfused through the pulsatile flow system, and adhesion on silicone catheters was recorded. These results were compared with those obtained under static cell culture conditions. Astrocyte adhesion was significantly increased under conditions of increased flow rate (0.25 and 0.30 mL/min), and a trend toward increased adhesion was observed under conditions of elevated pressure and pulsation rate. Because the HSCB represents physiologic conditions more accurately than static cell culture, our results suggest that standard static cell culturing techniques are insufficient to model inflammatory cell adhesion on catheters used in the treatment of hydrocephalus and that changes to the ventricular microenvironment can alter the mechanisms of cellular adhesion. The HSCB represents a relevant test system and is an effective model system for the analysis of cellular adhesion and occlusion of shunt catheters.

Anti-fouling phosphorylcholine bearing polyelectrolyte multilayers: Cell adhesion resistance at rest and under stretching

We investigate the anti-fouling properties of polyelectrolyte multilayers bearing phosphorylcholine and triethylene glycol moieties and their adhesive response under stretching towards mammalian cells and fungi. More precisely we use a precursor multilayer deposited on glass and on an elastomeric silicone sheet and onto which one or two layers of polyacrylic acid modified with triethylene glycol or phosphorylcholine groups are added. In previous studies, these architectures proved to be resistant to protein adsorption (A. Reisch, J. C. Voegel, E. Gonthier, G. Decher, B. Senger, P. Schaaf and P. J. Mesini, Langmuir, 2009, 25, 3610; A. Reisch, J. Hemmerle, J. C. Voegel, E. Gonthier, G. Decher, N. Benkirane-Jessel, A. Chassepot, D. Mertz, P. Lavalle, P. Mesini and P. Schaaf, J. Mater. Chem., 2008, 18, 4242.). Here we investigate the adhesion of mammalian cells (fibroblasts) and of fungi (Candida albicans) both at rest and under uniaxial stretching of the substrate. Two layers of these polyelectrolytes yield surfaces that are practically resistant to the adhesion of fungi and mammalian cells at rest. Under stretching of the substrate, fungi adhesion remains almost totally prevented at least up to a stretching degree of 1.5, while fibroblast adhesion remains only prevented up to a stretching degree of 1.2. Fibroblast adhesion starts to take place and increases when the substrate is further stretched. The onset of fibroblast adhesion under stretching is retarded for phosphorylcholine containing films compared to those that contain triethylene glycol. These systems thus provide a first example of surfaces that present excellent anti-fouling properties at rest and become specifically adhesive under stretching.

Dynamic control over cell adhesive properties using molecular-based surface engineering strategies

In complex organisms, cells are often dependent on their extracellular matrix (ECM) for structural integrity, the mechanical properties of tissues, and for signaled regulation of cellular processes including adhesion, migration, growth, secretion, gene expression and apoptosis. Achieving dynamic control, i.e. by using an external stimulus, over the interactions between cells and artificial interfaces holds considerable promise in tissue engineering, medicine, cell biology and immunology. For example, improved spatial control over cell-surface interaction is potentially useful in the design of cell-based screening devices. Dynamic control over SAMs for cell adhesion provides an additional handle to direct and study the attachment of cells to surfaces, e.g., in studying cell spreading from a predetermined pattern in order to screen the cytotoxicity of drug candidates. However, 'reversible' control of cell adhesion onto substrates is an area that is still in its infancy. In this critical review recent developments in cell adhesion of mammalian cells to SAM-modified surfaces, the physical properties of which can be controlled by an external stimulus, e.g. by light, electrochemistry, etc., are discussed (118 references).

Selective Immobilization of Peptides Exclusively via N-Terminus Cysteines by Water-Driven Reactions on Surfaces

Immobilizing peptides or proteins on bioinert surfaces enables the elucidation of ligand-receptor interaction in complex biological systems. Here, we report a highly chemoselective surface reaction that immobilizes peptides exclusively via N-terminus cysteine residue in a peptide. At pH 5.5, only N-terminus cysteines of peptides couple covalently with phenoxy amino squarate moieties presented on self-assembled monolayers (SAMs) of alkanethiols on gold films. The selectivity of this surface reaction can tolerate the presence of internal cysteines in close proximity to basic residues such as histidines. We demonstrated this selective surface reaction by mammalian cell adhesion and by SAMDI mass spectroscopy of the SAMs.

Randomly oriented, upright SiO2 coated nanorods for reduced adhesion of mammalian cells

Cell interactions with nanostructures are of broad interest because of applications in controlling tissue response to biomedical implants. Here we show that dense and upright SiO2 coated nanorods nearly eliminate cell adhesion in fibroblasts and endothelial cells. The lack of adhesion is not due to a decrease in matrix protein adsorption on the nanostructures, but rather an inability of cells to assemble focal adhesions. Using spatially patterned nanorods, we show that cells display a preference for flat regions of the surface. Our results support a model in which interfering with nanoscale spacing of ligated integrins results in reduced cell adhesion and subsequent cell death. We propose that dense monolayers of nanorods are a promising nanotechnology for preventing mammalian cell fouling of biomaterials.

Experimental and theoretical investigation of chain length and surface coverage on fouling of surface grafted polypeptoids

Numerous strategies exist to prevent biological fouling of surfaces in physiological environments; our strategy focuses on the modification of surfaces with poly-N-substituted glycine oligomers (polypeptoids). We previously reported the synthesis and characterization of three novel polypeptoid polymers that can be used to modify titanium oxide surfaces, rendering the surfaces resistant to adsorption of proteins, to adhesion of mammalian and bacterial cells and to degradation by common protease enzymes. In this study, we investigated the effect of polypeptoid chain length on the antifouling properties of the modified surfaces. For these experiments we used poly(N-methoxyethyl) glycines with lengths between ten and fifty repeat units and determined the influence of chain length on coating thickness and density as well as resistance to protein adsorption and cellular adhesion. Short-term protein resistance remained low for all polymers, as measured by optical waveguide lightmode spectroscopy, while fibroblast adhesion after several weeks indicated reduced fouling resistance for the polypeptoid-modified surfaces with the shortest chain length polymer. Experimental observations were compared to predictions obtained from a molecular theory of polymer and protein adsorption. Good agreement was found between experiment and theory for the chain length dependence of peptoid grafting density, and for protein adsorption as a function of peptoid grafting density. The theoretical predictions provide specific guidelines for the surface coverage for each molecular weight for optimal antifouling. The predictions show the relationship between polymer layer structure and fouling.

Chemical modification of reactive multilayered films fabricated from poly(2-alkenyl azlactone)s: design of surfaces that prevent or promote mammalian cell adhesion and bacterial biofilm growth.

We report an approach to the design of reactive polymer films that can be functionalized post-fabrication to either prevent or promote the attachment and growth of cells. Our approach is based on the reactive layer-by-layer assembly of covalently crosslinked thin films using a synthetic polyamine and a polymer containing reactive azlactone functionality. Our results demonstrate (i) that the residual azlactone functionality in these films can be exploited to immobilize amine-functionalized chemical motifs similar to those that promote or prevent cell and protein adhesion when assembled as self-assembled monolayers on gold-coated surfaces and (ii) that the immobilization of these motifs changes significantly the behaviors and interactions of cells with the surfaces of these polymer films. We demonstrate that films treated with the hydrophobic molecule decylamine support the attachment and growth of mammalian cells in vitro. In contrast, films treated with the hydrophilic carbohydrate d-glucamine prevent cell adhesion and growth almost completely. The results of additional experiments suggest that these large differences in cell behavior can be understood, at least in part, in terms of differences in the abilities of these two different chemical motifs to promote or prevent the adsorption of protein onto film-coated surfaces. We demonstrate further that this approach can be used to pattern regions of these reactive films that resist the initial attachment and subsequent invasion of mammalian cells for periods of at least one month in the presence of serum-containing cell culture media. Finally, we report that films that prevent the adhesion and growth of mammalian cells also prevent the initial formation of bacterial biofilms when incubated in the presence of the clinically relevant pathogen Pseudomonas aeruginosa . The results of these studies, collectively, suggest the basis of general approaches to the fabrication and functionalization of thin films that prevent, promote, or pattern cell growth or the formation of biofilms on surfaces of interest in the contexts of both fundamental biological studies and a broad range of other practical applications.

Hydroxyapitite-multiwalled carbon nanotubes nanocomposite for adhesion and electrochemical study of human osteoblast-like cells (MG-63)

A new biocompatible interface made up of hydroxyapitite-multiwalled carbon nanotubes (HA-MWCNTs) nanocomposite was developed for adhesion and electrochemical detection of human osteoblast-like cells (MG-63). The HA-MWCNTs nanocomposite was synthesized by self-assembling nano-hydroxyapitite onto multiwalled carbon nanotubes in solution, which was characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. The adhesion of the MG-63 on the nanocomposite film was studied by quartz crystal microbalance and cyclic voltammetry. The nanocomposites interface showed improved immobilization capacity for cells and good biocompatibility for preserving the activity of immobilized living cells. The living cells immobilized on an HA-MWCNTs modified glassy carbon electrode exhibited an irreversible anodic peak response being positively associated with the density and viability of cells, which was used to describe the cells growth and evaluate the effectiveness of antitumor drug 5-fluorouracil (5-FU) on the cells. The obtained 5-FU cytotoxicity results agreed well with those from conventional MTT assays. The highly biocompatible HA-MWCNTs nanocomposite is also expected to be an appropriate matrix for the electrochemical investigation of adhesion, proliferation, and apoptosis of many other relevant mammalian cells.

Molecular Gradients of Bioinertness Reveal a Mechanistic Difference between Mammalian Cell Adhesion and Bacterial Biofilm Formation

Chemical gradients play an important role in guiding the activities of both eukaryotic and prokaryotic cells. Here, we used molecularly well-defined chemical gradients formed by self-assembled monolayers (SAMs) on gold films to reveal that mammalian cell adhesion and bacterial biofilm formation respond differently to a gradient of surface chemistry that resists cell attachment. Gradient self-assembled monolayers (SAMs) consisting of two mixed alkanethiols were fabricated by differential exposure of the gold film to one alkanethiol, followed by soaking in another alkanethiol solution. A gradient in bioinertness that resisted cell attachment was created on SAMs from a gradient in the surface density of HS(CH2)11(OCH2CH2)3OH, backfilled with either HS(CH2)11OH or HS(CH2)11CH3. Measurements of the amounts of mammalian cells and bacterial biofilms on these gradient surfaces reveal that, for mammalian cells, a critical density of adhesion ligands from absorbed proteins on surfaces exists for supporting maximum adhesion and proliferation, whereas for the bacterium Escherichia coli , the amount of biofilm formed on surfaces increased linearly with the surface density of adhesive groups (methyl or hydroxyl groups) in different media. These results are consistent with mammalian cell adhesion requiring an anchorage via specific molecular recognitions and suggest that biofilms can form by immobilization of bacteria via nonspecific interaction between bacteria and surfaces.

Synthesis of a photoimmobilizable histidine polymer for surface modification

AbstractA novel photoreactive polymer with histidine polar groups was synthesized through the copolymerization of two types of methacrylic acid, one carrying histidine groups and the other carrying azidoaniline groups. The polymer was photoimmobilized on polyester disks for surface modification. The effect of the surface modification on the hydrophilic and biofouling properties was investigated. Static contact angle measurements showed that the polymeric surface was modified to be comparatively hydrophilic in the polymer‐immobilized region. Micropattern immobilization was carried out with a photolithographic method. Atomic force microscopy measurements showed that the polymer was formed on the disks in response to ultraviolet irradiation. Protein adsorption was reduced on the polymer‐immobilized regions, and in those regions, spreading and adhesion of mammalian cells were reduced in comparison with that in nonimmobilized regions. In conclusion, a novel histidine‐containing polymer was photoreactively immobilized on a conventional polymer surface, and it had reduced interaction with proteins and cells. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

The effects of cell culture parameters on cell release kinetics from thermoresponsive surfaces

The thermoresponsive properties of poly(N-isopropyl acrylamide) (pNIPAM) have led to its wide use in bioengineering applications, including the reversible adhesion of mammalian cells. The groups performing this research have used different solutions to initiate cell release and have varied the temperature of the solution during detachment. To our knowledge, there has been no direct correlation between the solution identity or temperature on the efficiency of cell release from pNIPAM films. In this work, we present a study of the effect of the solution type and temperature used to initiate detachment on the time required to achieve 100% detachment of bovine aortic endothelial cells (BAECs) from pNIPAM. The pNIPAM films used in this work were obtained using a novel technique using a spin-coated solution containing pNIPAM (spNIPAM). We found that the fastest, most reliable release of cells occurred below the LCST of the polymer at 4 degrees C in serum free media (SFM). As it is sometimes desirable to stop cell metabolism at the time of detachment (e.g., to "freeze" protein expression prior to subsequent analysis), the use of extremely cold SFM would be ideal in such cases.

SPR Sensing and Imaging of Mammalian Cell Adhesion and Cell Responses for Stimulants

not Available.

Micropatterned atmospheric pressure discharge surface modification of fluorinated polymer films for mammalian cell adhesion and protein binding

Micropatterning by an easily accessible atmospheric pressure discharge setup was performed on fluorinated polymer surfaces. Two conductively-coated glass slides are employed, together with the polymer foils to be modified and shadow masks for defining the microstructures. Surface angle measurements indicated the presence of charged groups in the surface of the fluoropolymer, enabling the growth of mammalian cells on arrays of spots with 600 μm diameter. XPS and FTIR spectra revealed the incorporation of oxygen in the surface, while the generation of aldehyde groups on the surface of fluorinated polymer films was demonstrated by selective coupling of fluorescence-labeled aminodextrane to the activated spots. The described method paves the way for producing protein microarray chips on flexible fluoropolymer substrates with standard laboratory equipment.

Quartz crystal microbalance and electrochemical cytosensing on a chitosan/multiwalled carbon nanotubes/Au electrode

Abstract The piezoelectric quartz crystal microbalance (QCM) was used to monitor the adhesion of mammalian cells on a chitosan (CS)/multiwalled carbon nanotubes (MWCNTs) composite modified gold electrode. The morphology and chemical properties of the CS/MWCNTs film were characterized with scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. The human breast cancer cells (MCF-7) were adhered to and grown on the CS/MWCNTs film modified gold surface or a net CS film modified gold surface, and the process of which was continuously monitored and displayed by changes of the resonant frequency (Δ f 0 ) and the motional resistance (Δ R 1 ) of the QCM. The attachment/spreading process of the MCF-7 cells on the QCM Au electrode decreased the Δ f 0 and increased the Δ R 1 simultaneously, implying rather complicated effects (simultaneous mass, viscoelasticity and probable surface-stress load) on the sensor surface. The attachment rate and viability of the cells when proliferating on the two surfaces were detected by the MTT assay. The presence and state of cells on the electrode surface were confirmed by the fluorescent microscopy. Cyclic voltammetry and electrochemical impedance spectroscopy of the ferricyanide/ferrocyanide couple were examined before and after the cell adhesion. All data showed that the cell adhesion and proliferation processes were more efficient on the biocompatiable nanocomposite surfaces. The cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment.

The importance of the Rcs phosphorelay in the survival and pathogenesis of the enteropathogenic yersiniae

The human-pathogenic yersiniae represent an ideal species group to study the evolution of highly virulent bacteria, with Yersinia pestis having emerged from the enteropathogen Y. pseudotuberculosis an estimated 20 000 years ago. Sequence data reveal that the Y. pestis genome is in the early stages of decay and contains hundreds of non-functioning pseudogenes, some of which may be important in the enteric lifestyle of Y. pseudotuberculosis. Bioinformatic analysis of pseudogenes from seven Y. pestis genome sequences identified rcsD as a gene disrupted early in the evolution of this organism. This phosphotransfer protein is part the of the Rcs phosphorelay, a two-component system present in the Enterobacteriaceae which has been shown to regulate the expression of capsular polysaccharide and other virulence determinants in several species including Escherichia coli and Salmonella. Using microarray analysis, we determined that the Y. pseudotuberculosis Rcs phosphorelay regulates the expression of 136 coding sequences, of which 60 % are predicted to affect the cell envelope. Several putative virulence determinants were identified as being regulated by this phosphorelay, along with proteins involved in biofilm formation, motility, mammalian cell adhesion and stress survival. Phenotypic assays on defined mutants confirmed a role for the phosphorelay in these processes in both Y. pseudotuberculosis and Y. enterocolitica.