Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: attachment strength of the diatom Navicula and the green alga Ulva

To understand the role of surface wettability in adhesion of cells, the attachment of two different marine algae was studied on hydrophobic and hydrophilic polymer surfaces. Adhesion of cells of the diatom Navicula and sporelings (young plants) of the green macroalga Ulva to an underwater surface is mainly by interactions between the surface and the adhesive exopolymers, which the cells secrete upon settlement and during subsequent colonization and growth. Two types of block copolymers, one with poly(ethylene glycol) side-chains and the other with liquid crystalline, fluorinated side-chains, were used to prepare the hydrophilic and hydrophobic surfaces, respectively. The formation of a liquid crystalline smectic phase in the latter inhibited molecular reorganization at the surface, which is generally an issue when a highly hydrophobic surface is in contact with water. The adhesion strength was assessed by the fraction of settled cells (Navicula) or biomass (Ulva) that detached from the surface in a water flow channel with a wall shear stress of 53 Pa. The two species exhibited opposite adhesion behavior on the same sets of surfaces. While Navicula cells released more easily from hydrophilic surfaces, Ulva sporelings showed higher removal from hydrophobic surfaces. This highlights the importance of differences in cell-surface interactions in determining the strength of adhesion of cells to substrates.     

Model films from native cellulose nanofibrils. Preparation, swelling, and surface interactions

Native cellulose model films containing both amorphous and crystalline cellulose I regions were prepared by spin-coating aqueous cellulose nanofibril dispersions onto silica substrates. Nanofibrils from wood pulp with low and high charge density were used to prepare the model films. Because the low charged nanofibrils did not fully cover the silica substrates, an anchoring substance was selected to improve the coverage. The model surfaces were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The effect of nanofibril charge density, electrolyte concentration, and pH on swelling and surface interactions of the model film was studied by quartz crystal microbalance with dissipation (QCM-D) and AFM force measurements. The results showed that the best coverage for the low charged fibrils was achieved by using 3-aminopropyltrimethoxysilane (APTS) as an anchoring substance and hence it was chosen as the anchor. The AFM and XPS measurements showed that the fibrils are covering the substrates. Charge density of the fibrils affected the morphology of the model surfaces. The low charged fibrils formed a network structure while the highly charged fibrils formed denser film structure. The average thickness of the films corresponded to a monolayer of fibrils, and the average rms roughness of the films was 4 and 2 nm for the low and high charged nanofibril films, respectively. The model surfaces were stable in QCM-D swelling experiments, and the behavior of the nanofibril surfaces at different electrolyte concentrations and pHs correlated with other studies and the theories of Donnan. The AFM force measurements with the model surfaces showed well reproducible results, and the swelling results correlated with the swelling observed by QCM-D. Both steric and electrostatic forces were observed and the influence of steric forces increased as the films were swelling due to changes in pH and electrolyte concentration. These films differ from previous model cellulose films due to their chemical composition (crystalline cellulose I and amorphous regions) and fibrillar structure and hence serve as excellent models for the pulp fiber surface.     

The role of conditioning film formation in Pseudomonas aeruginosa PAO1 adhesion to inert surfaces in aquatic environments

Bacterial initial adhesion to inert surfaces in aquatic environments is highly dependent on the surface properties of the substratum, which can be altered significantly by the formation of conditioning films. In this study, the impact of conditioning films formed with extracellular polymeric substances (EPS) on bacterial adhesion was investigated. Adhesion of wild type Pseudomonas aeruginosa PAO1 to slides coated with model EPS components (alginate, humic substances, and bovine serum albumin (BSA)) was examined. Surface roughness of conditioning film coated slides was evaluated by atomic force microscopy (AFM), and its effect on the bacterial initial adhesion was not significant. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrates. Results showed that bacterial adhesion to bare slides and slides coated with alginate and humic substances increased as ionic strength increased. Conversely, BSA coating enhanced bacterial adhesion at low ionic strength but hindered adhesion at higher ionic strength. It was concluded that forces other than hydrophobic and electrostatic interactions were involved in controlling bacterial adhesion to BSA coated surfaces. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces was shown to adequately describe the observed bacterial adhesion behaviors.     

Early adhesion of human mesenchymal stem cells on TiO(2) surfaces studied by single-cell force spectroscopy measurements

Understanding the interactions involved in the adhesion of living cells on surfaces is essential in the field of tissue engineering and biomaterials. In this study, we investigate the early adhesion of living human mesenchymal stem cells (hMSCs) on flat titanium dioxide (TiO(2) ) and on nanoporous crystallized TiO(2) surfaces with the use of atomic force microscopy-based single-cell force spectroscopy measurements. The choice of the substrate surfaces was motivated by the fact that implants widely used in orthopaedic and dental surgery are made in Ti and its alloys. Nanoporous TiO(2) surfaces were produced by anodization of Ti surfaces. In a typical force spectroscopy experiment, one living hMSC, immobilized onto a fibronectine-functionalized tipless lever is brought in contact with the surface of interest for 30 s before being detached while recording force-distance curves. Adhesion of hMSCs on nanoporous TiO(2) substrates having inner pore diameter of 45 nm was lower by approximately 25% than on TiO(2) flat surfaces. Force-distance curves exhibited also force steps that can be related to the pulling of membrane tethers from the cell membrane. The mean force step was equal to 35 pN for a given speed independently of the substrate surface probed. The number of tethers observed was substrate dependent. Our results suggest that the strength of the initial adhesion between hMSCs and flat or nanoporous TiO(2) surfaces is driven by the adsorption of proteins deposited from serum in the culture media.     

Bacterial adhesion: A physicochemical approach

The adhesion of bacteria to solid surfaces was studied using a physicochemical approach. Adhesion to negatively charged polystyrene was found to be reversible and could be described quantitatively using the DLVO theory for colloidal stability, i.e., in terms of Van der Waals and electrostatic interactions. The influence of the latter was assessed by varying the electrolyte strength. Adhesion increased with increasing electrolyte strength. The adhesion Gibbs energy for a bacterium and a negatively charged polystyrene surface was estimated from adhesion isotherms and was found to be 2–3 kT per cell. This low value corresponds to an adhesion in the secondary minimum of interaction as described by the DLVO theory. The consequences of these findings for adhesion in the natural environment are discussed.     

Adhesion of a <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain isolated from a paper machine to cellulose fibres

The adhesion to cellulose fibres of a strain of Pseudomonas putida isolated from a paper machine was studied under different environmental conditions. The physicochemical properties of both P. putida cells and cellulose fibres were also determined to better understand the adhesion phenomenon. Adhesion was rapid (1 min) and increased with time, cell concentration and temperature (from 25 to 40°C), indicating that bacterial adhesion to cellulose fibres is essentially governed by a physicochemical process. The P. putida cell surface was negatively charged, as shown by electrophoretic mobility measurements, and was hydrophilic due to a strong electron-donor character, as shown by the microbial adhesion to solvents method. Cellulose fibres were shown to be hydrophilic by contact angle measurements using the capillary rise method. These results suggest the importance of Lewis acid-base interactions in the adhesion process. In various ionic solutions (NaCl, KCl, CaCl₂ and MgCl₂), adhesion increased with increasing ionic strength up to 10–100 mM, indicating that, at low ionic strength, electrostatic interactions were involved in the adhesion process. An increase in the C/N ratio of the growth medium (from 5 to 90) decreased adhesion but this could not be related to changes in physicochemical properties, suggesting that other factors may be involved. In practice, temperature, ionic strength and nitrogen concentration must be taken into consideration to reduce bacterial contamination in the paper industry.     

Adhesion of Chlorella vulgaris to solid surfaces, as mediated by physicochemical interactions

Although adhesion of bacteria and yeast have been extensively studied by a wide range of experimental and theoretical approaches, significantly less attention has been focused on microalgae adhesion to solid materials. This work is focused on physicochemical aspects of microalgae adhesion. The results are based on experimental characterization of surface properties of both microalgae and solids by contact angle and zeta potential measurements. These data are used in modeling the surface interactions (thermodynamic and colloidal models) resulting in quantitative prediction of the interaction intensities. Finally, the model predictions are compared with experimental adhesion tests of microalgae onto model solids in order to identify the physicochemical forces governing the microalgae–solid interaction. The model solids were prepared in order to cover a wide range of properties (hydrophobicity and surface charge). The results revealed that, in low ionic strength environment, the adhesion was influenced mostly by electrostatic attraction/repulsion between surfaces, while with increasing ionic strength grew the importance of apolar (hydrophobic) interactions. The impact of solid surface properties on the degree of colonization by microlagae was statistically more significant than the influence of medium composition on cell surface of Chlorella vulgaris.     

Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces.

The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case.     

Osteoblast adhesion on poly(L-lactic acid)/polystyrene demixed thin film blends: effect of nanotopography, surface chemistry, and wettability

Biomaterial surface characteristics are critical cues that regulate cell function. We produced a novel series of poly(l-lactic acid) (PLLA) and polystyrene demixed nanotopographic films to provide nonbiological cell-stimulating cues. The increase in PLLA weight fraction (phi) in blend solutions resulted in topography changes in spin-cast films from pit-dominant to island-dominant morphologies having nanoscale depth or height (3-29 nm). Lower molecular weight PLLA segregated to the top surface of demixed films, as observed by X-ray photoelectron spectroscopy and secondary ion mass spectroscopy (SIMS). For phi > or = 0.5, the topmost film layer was predominantly filled with PLLA (>96% by SIMS at 20-A depth). Nanotextured substrata stimulated osteoblastic cell adhesion to a greater degree than did flat PLLA (phi = 1), and this effect was more pronounced for nanoisland (phi = 0.7 and 0.9) relative to nanopit topographies (phi = 0.5). Demixed films having relatively lower water contact angles generally enhanced cell adhesion and spreading. Our results reveal that cell adhesion is affected by surface chemistry, topography, and wettability simultaneously and that nanotextured surfaces may be utilized in regulating cell adhesion.     

Fluorine-free mixed amphiphilic polymers based on PDMS and PEG side chains for fouling release applications

Fluorine-free mixed amphiphilic block copolymers with mixtures of short side groups of polydimethyl siloxane (PDMS) and polyethylene glycol (PEG) were synthesized and studied for their ability to influence the surface properties and control the adhesion of marine organisms to coated surfaces. The settlement (attachment) and strength of adhesion of two different marine algae, the green seaweed Ulva and the diatom Navicula, were evaluated against the surfaces. It is known that hydrophobic coatings based on polydimethyl siloxane elastomers (PDMSe) are prone to protein adsorption and accumulation of strongly adherent diatom slimes, in contrast to PEG-based hydrophilic surfaces that inhibit protein adsorption and moderate only weak adhesion of diatoms. By incorporating both PDMS and PEG side chains into the polymers, the effect of incorporating both polar and non-polar groups on fouling-release could be studied. The dry surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The ability of these mixed amphiphilic polymers to reconstruct in water was examined using underwater bubble contact angle and dynamic water contact angle experiments. To understand more about surface reconstruction behavior, protein adsorption experiments were carried out with fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) on both dry and pre-soaked surfaces.     

Pectinolytic enzymes of oral spirochetes from humans.

The kinetics of adhesion of a Mycobacterium sp. to cellulose diacetate reverse-osmosis membranes is described. This Mycobacterium sp. (strain BT2-4) was previously implicated in the initial stages of reverse-osmosis membrane biofouling at a wastewater reclamation facility. Adhesion of BT2-4 cells to the cellulose diacetate membrane surfaces occurred within 1 to 2 h at 30 degrees C and exhibited saturation-type kinetics which conformed closely to the Langmuir adsorption isotherm (Pearson r correlation coefficient = 0.977), a mathematical expression describing the partitioning of substances between a solution and solid-liquid interface. This suggests that the cellulose diacetate membrane surfaces may possess a finite number of available binding sites to which the mycobacteria can adhere. Treatment of the attached mycobacteria with different enzymes suggested that cell surface polypeptides, alpha-1, 4- or alpha-1,6-linked glucan polymers, and carboxyl ester bond-containing substances (possibly peptidoglycolipids) may be involved in mycobacterial adhesion. The possible implication of these findings for reverse-osmosis membrane biofouling are discussed.     

Adhesion of Nongerminated Botrytis cinerea Conidia to Several Substrata

Conidia of the plant pathogenic fungus Botrytis cinerea adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of B. cinerea depends, at least in part, on hydrophobic interactions between the conidia and substratum.     

Human osteoblast cell spreading and vinculin expression upon biomaterial surfaces

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation.     

Substrate Hydrophobicity and Adhesion of Uromyces Urediospores and Germlings

Terhune, B. T., and Hoch, A. C. 1993. Substrate hydrophobicity and adhesion of Uromyces urediospores and germlings. Experimental Mycology 17, 241-252. Adhesions of urediospores and urediospore germlings of Uromyces appendiculatus, the bean rust pathogen, to various substrata was evaluated with regard to surface wettability. A range of surface wettabilities, or conversely hydrophobicities, was obtained by coating glass or quartz substrates with various organosilanes. Adhesion of urediospores or germlings was evaluated after the spore or germling laden-silanized surfaces were washed. Both urediospores and germlings adhered most tenaciously to surfaces with wettability ratings less than 30. Such surfaces were polystyrene and glass treated with dimethyldichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichorosilane, and diphenyldichlorosilane. The degree of germling contact to the various surfaces correlated closely with hydrophobicity and with the adhesion of germlings. Induction of appressoria on quartz substrates bearing inductive topographies (0.5-μm-deep grooves) was also closely associated with the degree of hydrophobicity.     

Fluorine-free mixed amphiphilic polymers based on PDMS and PEG side chains for fouling release applications

Fluorine-free mixed amphiphilic block copolymers with mixtures of short side groups of polydimethyl siloxane (PDMS) and polyethylene glycol (PEG) were synthesized and studied for their ability to influence the surface properties and control the adhesion of marine organisms to coated surfaces. The settlement (attachment) and strength of adhesion of two different marine algae, the green seaweed Ulva and the diatom Navicula, were evaluated against the surfaces. It is known that hydrophobic coatings based on polydimethyl siloxane elastomers (PDMSe) are prone to protein adsorption and accumulation of strongly adherent diatom slimes, in contrast to PEG-based hydrophilic surfaces that inhibit protein adsorption and moderate only weak adhesion of diatoms. By incorporating both PDMS and PEG side chains into the polymers, the effect of incorporating both polar and non-polar groups on fouling-release could be studied. The dry surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The ability of these mixed amphiphilic polymers to reconstruct in water was examined using underwater bubble contact angle and dynamic water contact angle experiments. To understand more about surface reconstruction behavior, protein adsorption experiments were carried out with fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) on both dry and pre-soaked surfaces.     

Morphology of modified regenerated model cellulose II surfaces studied by atomic force microscopy: effect of carboxymethylation and heat treatment

Model cellulose II surfaces with different surface charge have been prepared from carboxymethylated wood pulp. AFM tapping-mode imaging in air showed that the introduction of charged groups into the film does not appreciably change the surface morphology. However, after a mild heat treatment (heating at 105 degrees C for 6 h), an irreversible surface structure change, from near spherical-type aggregates to a fibrillar structure, was observed. This might be attributed to the formation of strong hydrogen bonds in the crystalline region of the films while the amorphous regions shrank upon drying. The suitability of these charged cellulose films for surface forces studies was also investigated. At pH below the pK(a) of the carboxyl groups present in the film, the interaction force could be fit by a van der Waals force interaction. At higher pH, the interaction was of a purely electrostatic nature with no van der Waals component observable due to the swelling of the surfaces.     

Variability of the Influence of Physicochemical Factors Affecting Bacterial Adhesion to Polystyrene Substrata

The role of electrostatic and hydrophobic interactions and solid and liquid surface tensions in the adhesion of four bacterial species (Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp., and Flexibacter sp.) to hydrophobic polystyrene petri dishes and to more hydrophilic polystyrene tissue culture dishes was investigated. The effect of electrostatic interactions was investigated by determining the effects of different electrolyte solutions on attachment to and of different electrolyte and pH solutions on detachment from the polystyrene substrate. The significance of solid and liquid surface tensions and hydrophobic interactions was investigated by measuring the effects of different surfactants (including a concentration series of dimethyl sulfoxide) on adhesion and detachment. Adhesion varied with bacterial species, substratum, and electrolyte type and concentration, with no apparent correlation between adhesion and electrolyte valence or concentration. The influence of different pH and detergent solutions on bacterial detachment also varied with species, substratum, pH, and detergent type; however, the greatest degree of detachment of all strains from the surfaces was produced by detergent treatment. The results suggest that adhesion cannot be attributed to any one type of adhesive interaction. There was some evidence for both electrostatic and hydrophobic interactions, but neither interaction could wholly account for the data.     

An in vitro model to evaluate cell adhesion to metals used in implantation shows significant differences between palladium and gold or platinum

Adhesion of tissue cells to metallic implants is a major factor that is important for proper tissue integration. Adhesion of Swiss mouse 3T3 fibroblasts to gold, platinum and palladium surfaces was investigated. Immunofluorescence staining for the integrin subunits alphav and beta1 and the focal contact protein vinculin revealed that cells growing on gold and platinum expressed many focal contacts. In contrast, cells on palladium surfaces had reduced numbers of focal contacts shown by vinculin staining and failed to demonstrate expression of alphav and beta1 in focal contacts. Spread cell area was also significantly reduced on palladium than on other surfaces suggesting that cells on palladium were more weakly attached. This may be due to either a different molecular composition of focal contacts in cells grown on palladium surfaces or unusual microstructural properties of the palladium surface. This model is useful to evaluate adhesion of cells to different metal surfaces.     

Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers.

Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.     

Yeast cell adhesion on oligopeptide modified surfaces

Self-assembling oligopeptides are novel materials with potential bioengineering applications; this paper explores the use of one of these oligopeptides, EAK 16 II, for modifying the surface properties of cell-supporting substrates. To characterize the surface properties, thermodynamic measurements of liquid contact angle and surface free energy were correlated to atomic force microscopy (AFM) observations. A critical concentration of 0.1 mg/ml was found necessary to completely modify the surface properties of the substrate with EAK 16 II. Adhesion of a yeast cell, Candida utilis, was modified by the coating of EAK 16 II on both hydrophobic (plastic) and hydrophilic (glass) surfaces: Cell coverage was slightly enhanced on the glass substrate, but decreased significantly on the plastic substrate. This indicates that the yeast cell adhesion was mainly determined via hydrophobic interactions between the substrate and the cell wall. However, on the EAK 16 II modified glass substrate, surface roughness might be a factor in causing a slightly larger cell adhesion than that on bare glass. The morphology of adhered cells was also obtained with AFM imaging, showing a depression at the center of the cell on all substrates. Small depressions on the oligopeptide-coated surfaces and plastic substrate may indicate good water-retaining ability by the cell. There was no apparent difference in cell adhesion and morphology among cells obtained from lag, exponential and stationary growth phases.