INTRODUCTION

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The prevention of contamination caused by pathogenic microorganisms during the manufacture, processing, and packaging of food is of considerable importance to public health and consequently is a major issue for industry (30). In particular, there is increasing concern associated with the contamination arising from bacterial biofilms which develop on materials used during food manufacture (6, 19, 40). These communities of bacteria, often embedded in a matrix of organic polymers exuded by the cells, can be extremely difficult to remove, and complete eradication of the pathogens is difficult, time-consuming, and expensive (27). In general, the formation of bacterial biofilms is believed to take place over at least three stages: a reversible adsorption step (26), primary adhesion of microorganisms to a surface, and colonization (9). The rates of these processes vary widely depending on the environmental conditions and the type of microorganisms, but the adhesion and colonization stages are considered to be relatively slow compared to the first step of cell adsorption (17). In principle, it should be possible to retard, if not prevent, the formation of biofilms on substrates by using materials to which bacteria cannot initially attach, and such a material or surface coating would be of considerable commercial interest (7). In practice, however, synthetic materials that are capable of preventing bacterial adsorption have proved rather elusive, despite a significant volume of research (8, 11). Properties of the substrate, such as hydrophobicity (32), hydrophilicity (23), steric hindrance (24), roughness (22), and the existence of a "conditioning layer" at the surface (1), are all thought to be important in the initial cell attachment process.

In addition, there are large discrepancies in the data available on the adhesion of microorganisms to various synthetic materials. These disparities are mainly due to the different experimental conditions and protocols used in different laboratories as well as to the fact that the substrates employed are sometimes incompletely characterized both in terms of their functionality, i.e., the actual chemistry of the groups present, and in terms of their density at the surface. Consequently, it is often difficult to draw a meaningful comparison between results reported by different authors, and no simple structure-function correlation has yet emerged (33). The objective of this investigation was to carry out a comprehensive comparative study of bacterial adsorption by using a broad range of surfaces with controllable and precisely defined chemistry, such that various chemical and physicochemical factors which may be important in defining the surface at the molecular level could be assessed for their effect on the adhesion of the representative food pathogens Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli.

	MATERIALS AND METHODS

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Chemicals. Poly(ethyleneoxide)-monomethylether (MeO-PEO) (M_n = 350, 500, 750, 2,000, and 5,000), 2-[2-(2-methoxy)ethoxy]acetic acid (MeO-PEO-3-CO₂H), 3-mercaptopropanoic acid, 2-aminoethanethiol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4,4'-azobis(4-cyanovaleric acid) (ACVA), 3-aminopropyltriethoxysilane (APTES), ethanoyl chloride, butanoyl chloride, octanoyl chloride, 2-ethylhexanoyl chloride, decanoyl chloride, oleoyl chloride, myristoyl chloride, phenolphthalein (A.C.S. reagent), hydrogen peroxide, triethylamine, toluene, 1,1,2-trichlorotrifluoroethane (A.C.S. reagent), tetrahydrofuran (THF), dansyl chloride, and sodium carbonate were purchased from Aldrich Chemical Company (Gillingham, United Kingdom). Perfluorobutanoyl chloride and perfluorooctanoyl chloride were purchased from Lancaster Synthesis (Lancaster, United Kingdom). Methanol (high-pressure liquid chromatography [HPLC] grade), 2-propanol (HPLC grade), acetone (AR grade), diethyl ether (HPLC grade, peroxide-free), sodium hydrogen carbonate, sodium hydroxide, potassium dichromate, sulfuric acid, and p-toluene sulfonoyl chloride were purchased from Fisher Scientific (Loughborough, United Kingdom). Azobis(isobutyronitrile) (AIBN) was purchased from Acros Organics (Loughborough, United Kingdom) and recrystallized from methanol prior to use. 2-Mercaptoethanol, ethanol (AR grade), and chloroform (AR grade) were purchased from BDH (Poole, United Kingdom). Sulfo-SDTB was purchased from Pierce (Rockford, Ill.). N-Methylacrylamide was purchased from Monomer-Polymer Laboratories (Trevose, Pa.). Krytox acid terminal perfluoropolyether was obtained from DuPont (Deepwater, N.J.). Cytochrome c (from bovine heart) and bovine serum albumin (BSA) were obtained from Sigma (Poole, United Kingdom). D-[U-¹⁴C]glucose and ³H-acetic anhydride were obtained from Amersham Life Sciences (Amersham, United Kingdom). Unless otherwise stated, all chemicals were used as received.

Growth and maintenance of microorganisms. Bacterial cultures were maintained on Tryptone Soya Agar (Oxoid) at 4°C. Stock cultures were grown statically overnight at 37°C in coryneform broth (containing [in grams per liter] tryptone, 10; yeast extract, 5; sodium chloride, 5; and glucose, 5; pH 7.2) for L. monocytogenes C200 type 2 and in yeast-dextrose broth (containing [in grams per liter] peptone, 10; beef extract, 8, sodium chloride, 5; glucose, 5; and yeast extract, 3; pH 6.8) for S. typhimurium LT2 50185, S. aureus NCDO 949, and E. coli NCFB 1989. Aliquots (100 ml) of stock culture in sterile Eppendorf tubes were drop-frozen in liquid nitrogen and stored at 70°C prior to use. Prior to the first adhesion experiments, growth curves were established for the bacteria by correlating plate counts and optical density measurements. Bacteria plated from the stationary phase showed no significant loss in viability after 24 h, as determined by plate counts. Cell viability after the adsorption experiments was qualitatively confirmed by using confocal microscopy, which clearly showed the formation of bacterial colonies on the grafted surfaces and plate counts, by using polymer beads rather than films but with the same surface functionality.

Preparation and characterization of silylated glass surfaces. Glass coverslips (13-mm diameter; Chance Propper, Ltd., Smethwick, United Kingdom) were hydrolyzed by immersion in sodium hydroxide (aqueous, 5 M) for 1 h and washed thoroughly with deionized water. They were then soaked in fresh piranha solution (70% sulfuric acid, 30% hydrogen peroxide) for 1 h, washed with water, and dried in air. The glass surfaces were prepared immediately prior to silylation with APTES by drying them in a hot oven for 30 min. The coverslips were then immersed in a solution containing 95% acetic acid (1 mM) in methanol, 4% water, and 1% APTES for 30 min at room temperature with occasional gentle shaking. The resultant amine-functional surfaces were finally washed with methanol (three times with 50 ml) and cured in a hot oven for 30 min.

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Synthesis of reagents. (i) MeO-PEO-CO₂H. Synthesis was carried out as described previously (13). The protocol described below is for MeO-PEO-5000, but a similar procedure was used for other PEO oligomers. High-molecular-weight MeO-PEO-OH (5.0 g, 1 mmol) was dissolved in anhydrous DMF (100 ml) with 1.1 eQ of succinic anhydride (0.1 g) in a dry, three-necked 250-ml round-bottomed flask and heated to 100°C under an atmosphere of dry nitrogen overnight. Upon cooling the mixture was concentrated under reduced pressure, and the polymer was purified by precipitation into cold ether (three times, 250 ml), followed by drying in a vacuum oven at 40°C overnight.

(ii) MeO-PEO acyl chlorides. High-molecular-weight carboxyl-terminated MeO-PEO (5.1 g, 1 mmol) was dissolved in dry toluene (100 ml) in a 250-ml three-necked round-bottomed flask. Oxalyl chloride (0.375 g, 3 mmol) was added, and the mixture was brought to reflux under an atmosphere of dry nitrogen and stirred at reflux overnight. Upon cooling the solvent and excess oxalyl chloride were removed under reduced pressure, and the resultant MeO-PEO-acyl chloride was used immediately. The acyl chloride of 2-[2-(2-methoxy)ethoxy]acetic acid (MeO-PEO-3-COCl) was synthesized in the same way.

(iii) Krytox acyl chloride. Krytox (5.0 g) was dissolved in 1,1,2-trichlorotrifluoroethane (100 ml) in a 250-ml three-necked flask. Oxalyl chloride (3 ml) was added, and the mixture was brought to reflux under an atmosphere of dry nitrogen and stirred at reflux overnight. Upon cooling the solvent and excess oxalyl chloride were removed under reduced pressure, and the resultant acid chloride was used immediately.

(iv) Poly(N-methylacrylamide) polymers. In a thick-walled tube, monomer (N-methylacrylamide, 10 g) was dissolved in 2-propanol (40 ml) with chain transfer agent (0.344 mmol) and initiator (AIBN or ACVA [see below], 2.83 mmol). This mixture was degassed by freeze-thaw cycles under vacuum at least three times. The tubes were then placed in a thermostatted oil bath at 65°C for 24 h. After it cooled to room temperature, the mixture was concentrated under reduced pressure, and the residue was added to diethyl ether (250 ml) to precipitate the polymer. This was filtered and the residue was redissolved in THF and reprecipitated into diethyl ether (three times, 250 ml) to leave the purified polymer as a colorless precipitate which was then dried in vacuo at 20°C overnight.

Chemical modification of surfaces. (i) Treatment with carboxyl-terminated poly(N-methylacrylamide). Silylated coverslips were placed in dilute hydrochloric acid (pH 6) and cooled to 0°C. Carboxyl-terminated poly(N-methylacrylamide) (1.0 g) was added to the buffer solution, followed by EDC (three times, 200 mg) every 30 min with gentle stirring. After the addition was complete, the surfaces were left at 0°C for a further 72 h. The surfaces were then rinsed repeatedly with deionized water (five times, 250 ml) and dried in air.

(ii) Treatment with acid chlorides. Glass coverslips were placed in dry chloroform (40 ml) and triethylamine (10 ml); to this the required acid chloride (5 ml) was added, and the mixture was left at room temperature under nitrogen with gentle stirring for 2 h. The coverslips were then washed with chloroform (four times, 50 ml) and dried in air.

(iii) Treatment with Krytox acid chloride. Glass coverslips were placed in 1,1,2-trichlorotrifluoroethane (10 ml) and triethylamine (3 ml) containing Krytox acid chloride (3 g) and left at room temperature with gentle stirring for 1 h; the coverslips were then washed in a Soxhlet apparatus with 1,1,2-trichlorotrifluoroethane overnight and dried in air.

Assessment of bacterial attachment to synthetic surfaces. To carry out radioactive labelling of microorganisms, modified coryneform broth (6 ml, containing a growth-limiting concentration of glucose [1 g · liter¹]) for cultures of L. monocytogenes or modified yeast-dextrose broth (6 ml, containing glucose [1 g · liter¹]) for cultures of S. typhimurium, S. aureus, or E. coli were inoculated with 50 µl of thawed stock culture. Aliquots of D-[U-¹⁴C]glucose were added to a final concentration of 20 µCi · ml¹ from a 1-mCi stock solution (230 to 370 mCi · ml¹; a sterilized, aqueous solution containing 3% ethanol). The cultures were incubated, statically, at 37°C for 24 h.

Determination of absolute cell counts. Aliquots (200 µl) of unlabelled bacterial culture were enumerated via a serial dilution method with Tryptone Soya Agar and incubation at 37°C for 24 h. The numbers of viable cells determined this way were compared with the scintillation counts from equivalent aliquots (200 µl) of radiolabelled bacteria. In addition, the scintillation readings were correlated with total bacterial numbers obtained via optical density measurements and microscopy.

Adsorption of proteins to synthetic surfaces. The method to prepare radioactively labelled proteins was adapted from that used by Freeman and Parish to label heparan (12). Cytochrome c or BSA (20 mg in each case) was dissolved in NaHCO₃ (aqueous 0.5 M, 1 ml) containing 10% (vol/vol) methanol in a sealed 15-ml reaction vial and cooled to 0°C in an ice bath. ³H-acetic anhydride (0.1 ml, 10 mCi; 500 mCi · mmol¹) in toluene was added, and the mixture was stirred for 3 h at 0°C. The mixture was acidified to pH 7.0 with acetic acid and allowed to warm to room temperature, and the toluene was removed under a stream of nitrogen. The solution was desalted by using a PD-10 column (Pharmacia Biotech, St. Albans, United Kingdom) equilibrated and developed with aqueous ethanol (10% [vol/vol]). Column fractions containing radioactive material appearing ahead of the ³H-acetate peak were pooled, sodium azide was added (0.1% [wt/vol]), and the solution was finally stored at 4°C. Protein concentration was determined by using the Lowry method for BSA and from a spectrophotometric calibration curve (408 nm) for cytochrome c.

Pretreatment of substrates with BSA and cell exudate. Native BSA (37.5 µg) was added to MOPS (50 mM, pH 7.0, 10 ml) in 15-ml screw-capped bottles. Glass coverslips (13-mm in diameter), previously stored in absolute ethanol, were transferred to the protein solutions. The bottles were incubated, with gentle shaking, for 1 h (unless otherwise stated) at 37°C. The coverslips were then rinsed in sterile MOPS (50 mM, pH 7.0, 10 ml) and transferred to 15-ml screw-capped bottles containing sterile MOPS (50 mM, pH 7.0, 10 ml). Aliquots (200 µl) of ¹⁴C-labelled L. monocytogenes suspension were added, and the bottles were incubated, with gentle shaking, for 24 h as described above. Glass coverslips were subsequently rinsed in sterile MOPS (50 mM, pH 7.0, 10 ml) and transferred to scintillation vials for counting by the standard method.

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Other analytical methods. Fourier transform-attenuated total internal reflection infrared spectra were recorded on a Perkin-Elmer 1600 spectrometer (Perkin-Elmer, Seer Green, United Kingdom) with a SpectraTech baseline attenuated, total internal reflectance apparatus by using a 45° germanium flat-face prism purchased from Nicolet Instruments (Nicolet, Warwick, United Kingdom). Fluorescence spectra were obtained by using a Perkin-Elmer LS50B Luminescence Spectrometer equipped with a front-face sample cell. Nuclear magnetic resonance (NMR) spectra were recorded on a Jeol-EX 270 NMR spectrometer (Jeol-UK, Welwyn Garden City, United Kingdom) by using CDCl₃ or CD₃SOCD₃ as the solvents, with residual proton signals as the internal reference. UV spectra of modified surfaces were recorded on a Perkin-Elmer Lambda 15 spectrometer.

	RESULTS

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Preparation and characterization of surfaces. Modified silica glass, in the form of 13-mm microscope coverslips, was chosen as a standard substrate for adhesion studies because of convenience of handling. As illustrated in Fig. 1, the coverslips were treated with APTES via the method of Durfor et al. (10) to give a slightly hydrophobic amine-functional surface, to which pendant groups of various degrees of chemical functionality, hydrophobicity, hydrophilicity, size, and hydrogen-bonding ability were grafted. Further derivatization reactions of APTES-modified coverslips were carried out by coupling acid chlorides or carboxylic acids to amine groups to generate short- and long-chain hydrophobic, hydrophilic, fluorocarbon, alkylamide, and alkylether surfaces. The functionalities of the surfaces grafted onto these SiO₂-APTES substrates are shown in Table 1.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Derivatization of glass substrates.

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View larger version (19K): [in this window] [in a new window]   FIG. 2.   (Top) Adsorption of BSA to functionalized surfaces. (Middle) On the left, kinetics of adsorption of BSA are shown: S-NH2 (), S-C6 (), and hydrophobized SiO2 (). On the right, the adsorption of BSA against concentration is shown: S-NH2 () and S-C8F (). (Bottom) Adsorption of cytochrome c to functionalized surfaces.

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Adhesion of microorganisms. Having established satisfactory protocols for surface modification and proven that the surfaces displayed sufficiently high density of the graft material even on a molecular scale, we proceeded to investigate the adhesion of microorganisms. L. monocytogenes, S. typhimurium, S. aureus, and E. coli were chosen as representative pathogens, although most of the initial experiments were conducted with L. monocytogenes. Interestingly enough, it was found that the pattern of L. monocytogenes adhesion was not too dissimilar from that of BSA. Once again, the greatest number of cells attached to hydrophobic substrates and the charged APTES surface. The hydrophilic surfaces of MeO-PEO-3 and MeO-PEO-5000 showed the highest resistance to cell attachment, according well with previous studies of these materials (25). As before, the acetamide graft surfaces also displayed low cell adhesion, although the long-chain analog poly(N-methylacrylamide) was less effective (Fig. 3). MeO-PEO-5000-modified substrates were found to be the least adsorptive for other bacteria, too, but the hydrophilic acetamide surface was less effective at preventing the adhesion of S. aureus and S. typhimurium than the low-surface-energy hydrophobic Krytox perfluoropolymer (Fig. 4). This result indicates that, for materials which are not particularly repellent for microorganisms, the surface chemistry of bacteria themselves plays an important role in the adsorption process. It is also worth noting that the kinetics of microorganism attachment were very different from those observed with proteins; in general, the number of cells bound to the substrates reached a stable level after 16 to 24 h of incubation compared to 1 h or less for proteins.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Adsorption of L. monocytogenes to functionalized surfaces.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Adsorption of microorganisms to selected surfaces: S-Krytox (open bars), S-C2 (shaded bars), and S-PEO-5000 (solid bars).

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Adsorption of L. monocytogenes to functionalized surfaces. The number of cells attached in the absence of BSA (open bars) and the number of cells attached to surfaces pretreated with BSA for 1 h (solid bars) are indicated.

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	DISCUSSION

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The prerequisite for our study of bacterial adhesion was the preparation and characterization of suitable, well-defined substrates such that the effects of physicochemical parameters, including hydrophobicity, hydrophilicity, steric hindrance, roughness, graft density, and functionality, on the adsorption phenomena could be assessed individually. The synthetic substrates (Table 1) were shown by microscopy and contact angle goniometry to be smooth over a "sub-bacterial" scale (i.e., <1 µm in length). Some evidence for surface rearrangements on APTES substrates was obtained during dynamic and equilibrium contact angle measurements, as judged by the hysteresis observed between the advancing and receding contact angles. The most probable explanation for this was that as the water drop advanced over the surface, amine groups were able to rearrange their conformation to point into the aqueous layer. Subsequent removal of the water drop left a more hydrophilic surface, which reverted to a more hydrophobic structure as amine groups rebound to the silica surface. However, all of the other substrates appeared to be stable and homogeneous at 37°C. The experimentally determined surface energies for derivatized substrates were as expected for materials with a high graft density, and this, combined with the microscopy (AFM and optical) and functional-group titration data, indicated that the substrates were chemically well defined and thus suitable as probes for studying adsorption phenomena.

Further evidence for the satisfactory surface density of the introduced functionality on a macromolecular scale came from the protein adsorption studies. BSA adsorption to the aminopropyl surfaces was close to the calculated figure for a monolayer (we assumed a surface area for BSA of 1.0 × 10¹⁶ m², based on approximate dimensions of 100 by 100 Å, and considered that no denaturation took place) and was essentially irreversible; increasing the amount of BSA in the medium did not lead to further adsorption. For cytochrome c the highest adsorption occurred to a hydrophobic uncharged surface and represented a coverage of ~90% of a monolayer, again assuming no denaturation of the protein took place on attachment. For "noncharged" alkylamide grafts, a clear correlation was obtained between the chain length of the amide and the numbers of protein molecules attached, with BSA adsorption reaching a maximum at alkyl chains containing six to eight methylene units. Thus, it appeared that with these and higher homologs, adsorption to what was effectively a hydrocarbon layer occurred. Attachment of the proteins to other hydrophobic substrates was as expected, with low-surface-energy perfluorinated materials displaying adsorption similar to that of the long-chain hydrocarbons.

The effect of grafting hydrophilic groups to the substrates was largely in accordance with previous studies (15). The hydrophilic MeO-PEO-5000 surface displayed the best protein-rejecting properties, and the short-chain MeO-PEO-3 also exhibited low protein adsorption (20, 18), although this latter surface proved less resistant to the smaller cytochrome c. The amine-terminal APTES surface might also have been expected to be hydrophilic under the experimental conditions (pH 7.0, 37°C); however, the surface energy recorded (34.76 mJ · m²) was similar to that of the longer-chain alkylamides. This suggested that at least some of the amine groups may have been "folded back" by binding to the siloxane surface, exposing the propyl chains to the solution (36). Reaction of the APTES substrates with acetyl chloride generated a more hydrophilic acetamide surface (39.38 mJ · m²), and this in turn reduced protein adsorption to levels similar to those of the MeO-PEO-3 substrates. The polymer analog of acetamide, poly(N-methylacrylamide) (PNMeAM) exhibited a rather higher degree of adsorption of BSA than did cytochrome c, which was not expected since it is hydrophilic, of high surface energy (40.66 mJ · m²), and might, in accordance with the mechanisms advanced for protein rejection by PEO, offer steric hindrance to attaching moieties via polymer chains extending into solution (indeed, MeO-PEO-5000 proved more resistant to cytochrome c adsorption than its short-chain counterpart MeO-PEO-3 in our experiments). It seems, therefore, that a simple combination of hydrophilicity and steric hindrance is not necessarily enough to reduce protein attachment, and the fact that MeO-PEO-5000 adsorbed far less BSA than PNMeAm perhaps indicates that it is the unique solution structure of PEO polymers (39), rather than their hydrophilicity and molecular weight, which accounts for their protein- and microorganism-rejecting abilities.

Bacterial adhesion was also lowest to the hydrophilic substrates MeO-PEO-3 and MeO-PEO-5000, although the numbers of cells attached to these substrates varied markedly between different microorganisms (e.g., 8.6 × 10⁴ cells · cm² for S. typhimurium on MeO-PEO-5000 and 2.3 × 10⁶ cells · cm² for S. aureus). In addition, there was a considerable difference in the attachment of different bacteria to the hydrophilic acetamide surface. Whereas the adhesion of L. monocytogenes and E. coli to acetamide followed a pattern similar to that of BSA and cytochrome c, attachment of S. typhimurium and S. aureus was higher to this substrate than to the hydrophobic Krytox surface. This may have been due to the latter bacteria exuding adhesion-promoting materials which adsorbed sufficiently to mask the underlying hydrophilic surface. In this case, adsorption of much less than a monolayer of exudate might result in sticky "patches" on the surface that are separated but within "bridging" distance for a cell. As a corollary, for the same hydrophilic acetamide surface and L. monocytogenes, preadsorption of BSA suppressed cell attachment considerably, even though the amount of BSA was ~10% of a monolayer.

Admittedly, such a consideration treats the microorganisms as "living colloids" (5), disregarding the specific roles of bacterial structures such as pili, cell wall components, and extracellular lipopolysaccharides which have been the subject of much work elsewhere by a number of groups (21, 28) and which are considered to be of particular importance in the later stages of biofilm formation. However, it has been argued by a number of authors that these structural features are of less significance in the initial stages of the attachment process than the intrinsic thermodynamic factors involved (4, 29), and a number of detailed studies have been carried out to support this assertion (37). Our study has shown that over the time period of protein adsorption and initial cell attachment (1 to 24 h), this assumption is reasonable, since the overall pattern of cell adhesion to different substrates was similar among the various cell types, although the absolute numbers varied considerably.

In conclusion, hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption and cell attachment, and a clear correlation between substrate chemistry and protein adsorption was established. In addition, our studies supported the view that the effectiveness of PEO polymers in preventing protein adsorption cannot be attributed directly to hydrophilicity and molecular weight alone. For our model systems, protein adsorption could be approximately correlated with short-term cell adhesion, and bacterial attachment for some microorganisms also correlated with substrate chemistry. However, for S. aureus and S. typhimurium a different mechanism of cell attachment appeared to occur, as shown by the relative numbers of cells adsorbed to hydrophobic and hydrophilic substrates. Preadsorption of BSA resulted in a reduction of cell adhesion to selected surfaces in 24-h assays. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups, if grafted uniformly and in sufficient density to the surface, may be the best method for preventing cell adsorption to synthetic substrates.