Biomaterial surface-dependent neutrophil mobility

Yue Zhou,1 Claire M. Doerschuk,2 James M. Anderson,1,3,4 Roger E. Marchant1,3

1Department of Biomedical Engineering, Case Western Reserve University, Wickenden Building, 10900 Euclid Avenue,Cleveland, Ohio 441062Department of Pediatrics, Rainbow Babies & Children’s Hospital, Cleveland, Ohio 441063Macromolecular Science, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 441064The Institute of Pathology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106

Received 12 November 2003; revised 28 January 2004; accepted 28 January 2004Published online 26 April 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30015

Abstract: Compromised neutrophil function in the pres-ence of an implanted biomaterial may represent an impor-tant mechanism that allows for the development of implant-associated infections. Here, human neutrophil mobility hasbeen investigated on a polyurethane (ChronoFlex AR), ahydrophobic surface consisting of an octadecyltrichlorosi-lane (OTS) self-assembled monolayer, and a glass referencematerial. Neutrophil mobility was quantified, based on cellmovement speed and persistence time obtained from time-lapse optical microscopy, while neutrophil cytoskeletalstructures and morphology were visualized using confocalmicroscopy and atomic force microscopy. Our results showthat material surface properties affect neutrophil–surfaceinteractions, as reflected by morphological changes, and the

mobility of neutrophils stimulated by N-formylmethionyl-leucyl-phenylalanine (fMLP). In the absence of adsorbedplasma proteins, the mobility of stimulated neutrophils in-creased with increasing material hydrophobicity from glass,to polyurethane, to OTS. The opposite trend was observed inthe presence of adsorbed plasma proteins, such that neutro-phil mobility increased with decreasing material hydropho-bicity. Analysis of the results showed that the mobility offMLP-stimulated neutrophils cells was inversely related tothe extent of cell spreading on the materials. © 2004 WileyPeriodicals, Inc. J Biomed Mater Res 69A: 611–620, 2004

Key words: neutrophils; mobility; biomaterials; AFM; timelapse

INTRODUCTION

A variety of cardiovascular devices have been de-veloped to assist in the performance of importantphysiological functions. However, device-associatedinfection is a serious complication, which occurs de-spite intrinsic host defense mechanisms. Even thoughthe incidence of cardiovascular device-associated in-fection is low, the associated morbidity and mortalityis significant.1,2 Once an infection occurs, the implantoften has to be removed to eliminate the complica-tion.3 The mechanisms underlying the device-associ-ated infections are not well understood, although thepresence of an implanted material may contribute toreduced effectiveness of the host defense systems. Therole of the biomaterial is an important factor, because

the number of organisms required to initiate an infec-tion is reduced in the present of an implant.4,5 Inaddition, some normally nonpathogenic bacteria, suchas Staphylococcus epidermidis, which are eliminated ef-ficiently by host defense mechanisms, are frequentlyidentified as the infecting organism in cardiovasculardevices.6,7 One of the most important virulent factorsassociated with S. epidermidis and other bacteria is theformation of a biofilm on the implant surface. A bio-film is believed to protect bacteria from attack by hostphagocytic cells.7,8

Neutrophils are the predominant cell type in thefirst 1–2 days of the acute phase inflammatory re-sponse, and are the first cells to arrive at the site of aninfection.9 In addition, neutrophils are crucial in thehost’s defense against invading bacteria. Therefore,neutrophils represent the earliest, and possibly thebest, opportunity to eliminate bacteria from a bioma-terial surface before biofilm formation occurs. In re-sponse to infection, neutrophils migrate to the bacteriaby detecting chemoattractants released from the infec-tion site, and then eliminate the bacteria through themechanism of phagocytosis. If neutrophil mobility isreduced on a biomaterial surface, this may interfere

Correspondence to: Roger E Marchant, e-mail: rxm4@po.cwru.edu

Contract grant sponsor: the National Institutes of Health;contract grant number: EB-00279

Contract grant sponsor: the Center for CardiovascularBiomaterials at CWRU

© 2004 Wiley Periodicals, Inc.

with neutrophil access to the bacteria, and give thebacteria sufficient opportunity to produce a biofilm.Previous studies have shown that biomaterials mayaffect neutrophil mobility,10 but the mechanism is stillnot well understood.

In this report, human neutrophil mobility and mor-phology on a clinical polyurethane biomaterial and onmodel surfaces were investigated using in vitro time-lapse optical microscopy, atomic force microscopy(AFM), and laser confocal microscopy. To quantify therole of adsorbed plasma proteins on neutrophil mo-bility, the materials were studied in the presence andabsence of human plasma proteins surface-adsorbedfrom platelet poor plasma (PPP). The results show thatneutrophil mobility and morphology are closely re-lated, and are dependent on the surface properties ofthe biomaterials, and on the availability of surface-adsorbed plasma proteins.

MATERIALS AND METHODS

Material surface preparation

Neutrophil mobility was investigated on three materialsurfaces: a clinically relevant polyurethane (ChronoFlex AR,Cardio Tech International, Inc), a hydrophobic model sur-face consisting of an octadecyltrichlorosilane (OTS) self-as-sembled monolayer (SAM), and glass coverslips. A polyure-thane was chosen, because it is one of the most broadly usedpolymeric biomaterials in cardiovascular devices. The OTS-SAM surface, which has terminal methyl groups, was cho-sen as a model surface for hydrophobic polymeric biomate-rials used in cardiovascular devices, such as siliconeelastomer or polyethylene. Glass coverslips were chosen as ahydrophilic reference material. Therefore, these three mate-rials provide a set of broad changes in surface propertiesranging from the hydrophobic to the hydrophilic.

The polyurethane and OTS-SAM were prepared on glasscoverslips. The glass coverslips were cleaned by soaking inwarmed (�70°C) chromic acid solution for 30 min, followedby rinsing with distilled water (Millipore Milli-Q� UV Plussystem, Millipore, Bedford, MA) and drying in an oven at�90°C. Glass coverslips cleaned by this procedure wereused as the hydrophilic reference material. The polyure-thane surfaces were prepared by coating a thin layer ofpolyurethane solution directly on glass coverslips. Medical-grade aromatic, polycarbonate-based polyurethane solution[22% in dimethyl acetamide (DMAC), ChronoFlex AR, Car-dio Tech International, Inc.] was diluted to a concentrationof 1% (wt) by DMAC (99�% pure, Sigma Chemical Co.). Thesolution was deposited on each coverslip and allowed tospread over the surface. The coverslips were then dried in anoven at 65°C for over 4 h to eliminate residual solvent. Thisprocedure provided a smooth polyurethane coating on theglass. A reproducible water contact angle of 73 � 1°, wasused to confirm the quality of the polyurethane coating.

The hydrophobic OTS SAM surface was prepared by de-positing octadecyltrichlorosilane (Aldrich Chemical, Mil-

waukee, WI) on glass coverslips from dicyclohexyl solution.Both OTS and dicyclohexyl (Aldrich Chemical) were vac-uum distilled before use. Briefly, cleaned glass coverslipswere subjected to an argon/H2O a rediofrequency glow-discharge treatment under 0.5 mmHg pressure for 15 min, aprocedure that increases surface hydroxyl groups for silanecoupling. The coverslips were then placed in dicyclohexylcontaining OTS (2.5 mM) for 30 min on each side. The OTSmodified coverslips were later rinsed and sonicated inHPLC grade chloroform for 20 min, followed by a secondrinse and brief air drying. The OTS SAM provides a surfacewith uniform terminal methyl groups, which creates asmooth, highly hydrophobic, surface. A reproducible watercontact angle of 109 � 1° was used to confirm the quality ofthe OTS monolayer.

Neutrophil and plasma isolation

Human neutrophils were isolated from nonmedicatedblood donors. Sodium citrate (Sigma Chemical Co., St.Louis, MO) was used as anticoagulant. Neutrophil isolationwas performed using density gradient centrifugation in Fi-coll-Paque (Pharmacia Biotech, Uppsalla, Sweden), and sed-imentation in Dextran T500 (pH 7.4; Pharmacia Biotech),followed by hypotonic cell lysis of residual red blood cells.The neutrophils obtained were �95% viable, and were sus-pended in Hanks’ balanced salts (HBSS, Sigma ChemicalCo.) without Ca2� or Mg2� to a concentration of 4 � 106

cell/mL as a stock suspension. Platelet-poor plasma (PPP)was isolated from the blood of the same donor. The wholeblood, anticoagulated with sodium citrate, was centrifugedat 1100 � g to obtain PPP. The PPP was then diluted withphosphate-buffered saline (PBS) so that the final concentra-tion of plasma in PBS was 10% by volume.

Time-lapse optical microscopy

Neutrophil movement on the material surfaces was re-corded using a time-lapse optical microscopy. Digitized im-ages were collected at 30-s intervals using a computer-con-trolled digital image capture/processing system linked to aNikon Diaphot 200 inverted optical microscope (with LWD40� objective lens). Test substrata were mounted in a flowcell system, which was filled with HBSS, and then replacedby a neutrophil suspension (4 � 105 cell/mL) in HBSS,diluted from a stock suspension and supplemented withCa2� or Mg2� just before the experiment. The system wasincubated at ambient temperature for 5 min to allow time forneutrophil adhesion. The neutrophil suspension was thenexchanged with HBSS to remove nonadherent neutrophils.The cell movement was then recorded using the time-lapsemicroscopy.

To study neutrophil responses in the presence of a che-moattractant, N-formylmethionyl-leucyl-phenylalanine (fMLP)was used. fMLP is the most commonly used activator forneutrophils in vitro. After neutrophil adhesion, the mediumwas exchanged with fMLP (10�8 M) solution in HBSS. Pub-lished reports indicate that a fMLP concentration of 10�8 M

612 ZHOU ET AL.

facilitates optimal cell migration speed and polarization.11,12

Time-lapse optical microscopic images were collected at am-bient temperature over a period of 30 min.

For examination of neutrophil movement on the materialsin the presence of adsorbed plasma proteins, the flow cellwas first filled with PBS, which was then replaced with 10%PPP. The system was then incubated for 15 min, followed byexchange with PBS and then HBSS. The same procedure forneutrophil locomotion as described above was then fol-lowed.

After image collection, the movement of neutrophils wasfollowed by scrolling through the images collected in atime-lapse stack. The cell velocity, and rate of directionalchange, during neutrophil movement, were computed fromthe changes in their positions in each sequential image, asdescribed under data analysis.

Neutrophil spread areas were measured from the contourline of each cell obtained from the phase contrast micros-copy images, using Scion Images software (Scion Copora-tion, Frederick, MD). Cell spreading areas from at least 30cells on each surface were analyzed, and used to comparebetween different surfaces.

Confocal laser-scanning microscopy

A Zeiss, LSM 410 laser scanning confocal microscope (Ar-gon/Krypton laser with excitation lines of 488 and 568 nm)was used to obtain fluorescence images of neutrophil cy-toskeletal morphology. The same procedures as describedfor time-lapse optical microscopy were followed to preparethe samples. Fifteen minutes after neutrophil adhesion orfMLP stimulation, the cells were fixed with 1% paraformal-dehyde (PFA) in PBS. Then, staining agent containing FITC–phalloidin (Sigma Chemical Co.), and Ethidium Bromide(2,7-Diamino-10-ethyl-9-phenyl-phenanthridinium bromide,Sigma Chemical Co.) were added to stain the cell F-actin andnuclei, respectively. L-�-Lysophosphatiycholine was addedto the staining solution to permeablize the cell membrane.Images were obtained using a 100� objective oil lens. Thefull width at half-max (FWHM), which represents the verti-cal resolution, was �0.7 �m. Images were collected at dif-ferent z-level to obtain the 3D organization of the F-actin.

Atomic force microscopy (AFM)

A Nanoscope IIIA Bioscope AFM (Digital Instruments)mounted on a Nikon Diaphot inverted microscope was usedto obtain tapping mode AFM images of neutrophil morphol-ogy on each material surface, in the absence of adsorbedprotein and fMLP. A glass coverslip coated with a testmaterial was mounted in a Bioscope fluid cell system, andHBSS was added into the fluid cell. A neutrophil suspensionwas then added into the fluid cell to a final neutrophilconcentration of 1 � 106 cell/mL. After a 5-min incubation,nonadherent cells were removed by fluid exchange withHBSS. Imaging was performed at ambient temperature us-ing a silicon nitride (Si3N4) tip. Cells were imaged at ascanning rate of 0.5 Hz. To obtain time series images, the

parameters for AFM imaging were kept constant duringrepeated scanning of the same area.

Data analysis

Random neutrophil movement can be characterized fromthe speed of locomotion and the frequency or rate withwhich the cell turns.13,14 The relative position of a neutrophilin each image frame was determined by tracking the centerpoint of the cell, as defined by the contour line of the cell.The centroid tracks were obtained by connecting the cellpositions in consecutive frames. The distance of locomotion() and angle of turn () in consecutive time intervals ofequal duration (�) was measured from the cell trajectory.13 Amathematical model, described by Dunn,14 was used tocharacterize the cell’s locomotion speed, and the ability tomaintain a specific direction during locomotion. The rootmean square (RMS) of the cell’s locomotion speed (S) duringeach step was calculated from:

S ��2�1/2

�(1)

The directional persistence time (P) was used to describecell’s ability to maintain a specific direction during locomo-tion.

P �2�

�2� (2)

Here, � is the time interval for collecting each consecutiveimage for all the experimental conditions. An increase ineither one of these two parameters corresponds to an in-crease in the rate of surface translation of the cells or mobil-ity in longer time scale.14,15

Statistically significant differences (p � 0.05) between mul-tiple means were determined by the method of one-wayanalysis of variance (ANOVA). Additionally, the two-tailedt-test was utilized to determine statistically significant dif-ferences (p � 0.05) between pairs of means. All statisticalanalysis was completed using software Origin 6.0.

RESULTS

Neutrophil mobility on material surfaces

Neutrophil mobility on each of the three materialssurface was studied under four conditions: with andwithout adsorption of plasma proteins, and with orwithout fMLP (10�8 M) stimulation. Neutrophil loco-motion (RMS speed) and direction persistence timeswere calculated from the cell trajectory using Equa-tions (1) and (2).

Neutrophil trajectory data, with fMLP stimulation,are illustrated in Figure 1(A) and (B). Cell trajectorieson the same material are plotted using the same initiallocation, and shifted for each material on the plots.

BIOMATERIAL SURFACE-DEPENDENT NEUTROPHIL MOBILITY 613

Without plasma protein adsorption [Fig. 1(A)], neu-trophil movement on glass was much less than onpolyurethane or OTS, while this order was reversed inthe presence of plasma protein adsorption [Fig. 1(B)].Notice also the large difference in distance scales forthe two conditions. The cell movement scales (�m)with plasma protein adsorption, are 10 times greaterthan without adsorbed plasma protein.

Neutrophils had low mobility on all three surfaces

under the negative control condition of no adsorbedproteins or fMLP stimulation. RMS speeds (Fig. 2)were all less than 2 �m/min, and persistence times(Fig. 3) were less than 0.4 min for these negativecontrols. The addition of fMLP (10�8 M) stimulationsignificantly increased neutrophil RMS speed and per-sistence time on the polyurethane (p � 0.01) and onthe OTS (p � 0.01), but not on the glass.

When plasma proteins were included, neutrophil

Figure 1. Trajectory examples of neutrophils moving on glass, polyurethane, and OTS SAM surfaces. The trajectories ofneutrophils on the same material are plotted from the same initial location. (A) Without plasma protein adsorption. (B) Withplasma protein adsorption. The total time was 10 min. Time interval for each step was 30 s.

Figure 2. Root-mean-square (RMS) speeds of neutrophils on glass, polyurethane, and OTS surfaces. The data show theaverage speed and stand error (n � 20).

614 ZHOU ET AL.

mobility (RMS speed) increased on all three surfaces (p� 0.01) compared with the negative control conditionof no adsorbed proteins or added fMLP (Fig. 2). Thehighest cell mobility (4–7 �m/min) was obtained un-der the conditions of fMLP stimulation and adsorbedproteins. Cell mobility on all three surfaces increasedsignificantly compared with the negative control con-ditions, and followed the order of glass polyure-thane � OTS. In addition, cell mobility on the poly-urethane and the glass increased significantly comparedwith adsorbed proteins or fMLP stimulation alone.This material dependent order also corresponded todramatic increases in persistence times obtained forcell on the glass and polyurethane surfaces (Fig. 3).

Neutrophil morphology on material surfaces

Laser confocal microscopy was used to analyze pos-sible effects of material surface properties on neutro-phil cytoskeletal structures. Images were collected atdifferent z-levels to obtain the 3D distribution of theF-actin and nucleus. The images shown in Figures 4and 5 were obtained from the region of the cell closestto the material surface.

The neutrophils displayed distinct cytoskeletal mor-phology on the material surfaces without any proteinadsorption or fMLP stimulation. Neutrophils werewell spread on the glass surface with broad uropods[Fig. 4(a)]. Based on the analysis of confocal images atdifferent z-levels, most of the F-actin formed very

close to the surface adhesion region. In addition, thecytoskeletal organization showed asymmetry, withgreater clustering of F-actin located at the leading endof the cell, and more diffuse at the uropods, as shownby the white arrow in Figure 4(a). However, the rela-tive density of F-actin at both ends of the cells werecomparable. No difference was found in cytoskeletalorganization in the presence of fMLP, except for arelative increase in the density of F-actin [Fig. 4(d)].

On the polyurethane [Fig. 4(b)], neutrophils wereless spread and more polarized, compared with cellson glass. The cytoskeleton at the tailing cell endformed long, narrow, and branched tails. The highestdensity of F-actin was found in the cell adhesion re-gion, but was less clustered, compared with the cellson glass. After fMLP stimulation [Fig. 4(e)], a generalincrease in the density of F-actin was observed, par-ticularly in the level of cell body away from the sub-stratum.

Neutrophils on the OTS surface [Fig. 4(c)] exhibitedless F-actin at the cell adhesion region and little evi-dence for extended tail-end structures, compared withcells on the polyurethane or glass. The inclusion offMLP [Fig. 4(f)] stimulated an increase in F-actin at theadhesion region, making the cell morphology moresimilar to cells on the polyurethane and glass. How-ever, F-actin clustering was not detected in the celladhesion region, and cell tails as seen on the polyure-thane were not evident.

The presence of adsorbed plasma proteins reducedneutrophil spreading significantly on the glass and

Figure 3. Persistence times of neutrophil movement on glass, polyurethane, and OTS surfaces. The data show the averagepersistence time and stand error (n � 20).

BIOMATERIAL SURFACE-DEPENDENT NEUTROPHIL MOBILITY 615

polyurethane, but not on the OTS [Fig. 5(a–c)]. MostF-actin was distributed around cell nuclei and awayfrom cell adhesion region. In addition, cell nuclei weremore condensed, compared with cells on materialswithout adsorbed proteins. With fMLP stimulation,neutrophil spreading and F-actin density increased forcells on all three materials [Fig. 5(d–f)].

Neutrophil spreading (Table I) on the three surfaceswas quantified from the measured cell areas obtainedfrom the phase contrast images. Cell spreading wastypically much higher on surfaces without adsorbedprotein and followed the order of glass � polyure-thane � OTS. FMLP stimulation increased cell spread-ing on all three surfaces. In the presence of adsorbedproteins, cell speading on the three materials wassimilar, which again increased with fMLP stimulation.However, under these conditions, the order is re-versed with cell spreading on OTS � polyurethane �glass.

Qualitatively, there appeared to be a correlationbetween cell spreading and cell (RMS) speed. Thisobservation was compared statistically and a good

correlation was obtained (F � 0.01) using linear re-gression analysis (Fig. 6). Neutrophils maintained thiscorrelation in the presence or absence of adsorbedplasma proteins, but not in the absence of fMLP stim-ulation.

To obtain complementary information, neutrophilmorphology also was examined by fluid tappingmode AFM (Fig. 7). Time series AFM images were ofneutrophils obtained on each material without ad-sorbed proteins or fMLP stimulation. On the glass[Fig. 7(a–c)], cells moved slowly with trailing broadflattened uropods [white arrow in Fig. 7(a)]. The uro-pods became remained stationary, while the cell bodymoved forward. Other AFM images (data not shown)showed cells releasing their uropods from the substra-tum, but leaving behind significant amounts of celldebris that traced out the uropod outline. On thepolyurethane [Fig. 7(d–f)], the cells showed significantsurface translocation over the imaging time. A rela-tively small uropod is shown at the rear of the cell inFigure 7(e). The uropod moved forward with the cellbody, but became highly branched and leaving a cell

Figure 4. Confocal microscopic images of neutrophils F-actin (green) and nuclei (red) on glass (a,d), polyurethane (b,e) andOTS SAM (c,f). The F-actin cytoskeleton and nuclei are labeled with FITC-phalloidin and ethidium bromide, respectively. Theimage a,b,c were obtained in the absence fMLP. The image d,e,f, were obtained in the presence of fMLP (10�8 M) stimulation.[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

616 ZHOU ET AL.

trace [white arrow in Fig. 7(f)]. On the OTS, the neu-trophil at the top of the image moved forward quickly,and the rear of the cell had smaller uropods comparedwith cells on other surfaces [white arrow in Fig. 7(h)].After forward movement, the uropod retracted fromthe substrate, without leaving any detectable cell trace.

Figure 5. Neutrophil cytoskeleton and nuclei on plasma protein adsorbed glass (a,d), polyurethane (b,e) and OTS SAM (c,f)surfaces. The image a,b,c were obtained without fMLP, while the image d,e,f, were obtained in the presence fMLP (10�8 M)stimulation. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

TABLE INeutrophil Spreading Area on Glass, Polyurethane, and

OTS surfaces

Media Conditions

Neutrophil Spreading Area (�m2)a

Glass Polyurethane OTS

Buffer† only (control) 190 � 5 138 � 12 117 � 5� fMLP 216 � 11 145 � 7 132 � 4� proteins 79 � 11 88 � 7 83 � 4� proteins, � fMLP 95 � 3 112 � 3 136 � 6

aData obtained from phase contrast optical microscopy.†Buffer media: HBSS.Spreading data (mean � standard deviation). Sample size:

n � 30.

Figure 6. The correlation of neutrophil spread area withthe cell RMS speed on glass, polyurethane, and OTS surfacesin the presence of fMLP (10�8 M) stimulation. The error barsshow the standard error of mean value. (E) Cells in theabsence of plasma protein adsorption; (�) cells in the pres-ence of plasma protein adsorption.

BIOMATERIAL SURFACE-DEPENDENT NEUTROPHIL MOBILITY 617

DISCUSSION

The goal of this study was to examine the effect ofbiomaterial surface properties on the locomotion abil-ity of surface adhered neutrophils. For this purpose,we compared neutrophil mobility and morphology onthree materials with contrasting surface properties(OTS, polyurethane, and glass). Effective neutrophilmobility should facilitate access to bacteria, preventbacteria from developing a protective biofilm, anddecrease the probability for an implant-related infec-tion. Conversely, evidence for decreased neutrophilmobility may represent an important factor that di-minishes effective recognition and phagocytosis ofbacteria.

Neutrophil locomotion is a spatially and a tempo-rally coordinated process that includes extension ofprotrusions, formation of protrusion-substratum at-tachments, cytoskeletal contraction, and detachmentof the cell from substratum at the tail end.16,17 Cellmobility can be rate-limited by either one or a combi-nation of these mechanistic steps, depending on theenvironmental conditions and the nature of cell-sub-stratum interactions.18,19 We examined the role of bothadsorbed plasma proteins and neutrophil stimulation(by fMLP), in an attempt to provide some insight intomaterial-dependent neutrophil behavior.

Overall, neutrophil mobility was increased by fMLPstimulation or by the presence of adsorbed plasmaproteins. However, evidence for material dependent

Figure 7. Time series AFM amplitude images of living neutrophils on glass (a–c), polyurethane (d–f), and OTS SAM (g–i)surfaces. Images were obtained by fluid tapping mode AFM in HBSS.

618 ZHOU ET AL.

neutrophil mobility became apparent only after fMLPstimulation, whether or not adsorbed proteins werepresent. In the absence of fMLP stimulation or ad-sorbed proteins, there was little difference in neutro-phil mobility on the three surfaces, but there weresubstantial differences in cell morphology. Cytoskel-etal structures and its clustering in the neutrophil–surface adhesion region are indicative of enhanced celladhesion,20 and increased in the order of glass �polyurethane � OTS. The concomitant increases incell spreading, obtained from phase contrast images,and the residual cell traces after tail detachment, ob-served by AFM, are consistent with these inferreddifferences in cell adhesion.20 Such morphological dif-ferences would be expected to correlate with neutro-phil mobility. For example, cell traces have been re-lated increased cell adhesion and retarded speed forstimulated cells.17,18,20,21 However, in a moving cell,both forward protrusion extension and detachment ofthe cell tail contribute to the overall cell locomotionrate.17 The absence of differences in cell mobility forunstimulated cells would then suggest that the gener-ation of forward protrusions by the neutrophils isreduced, which would limit cell locomotion. Underthis condition, material dependent differences in de-tachment of the neutrophil tail end and cell spreadingare not indicative of cell mobility. This suggestion alsowould apply to cell mobility on the three materialsdetermined in the presence of adsorbed plasma pro-teins.

Differences in cell mobility became apparent oncethe neutrophils were stimulated by fMLP, both in theabsence or presence of adsorbed proteins. Neutrophilmorphology and cytoskeletal structures at the cell ad-hesion region on each surface before and after fMLPstimulation were similar. After fMLP stimulation,however, the extent of cell spreading and residualtraces from cell tail detachment became inversely re-lated to cell mobility, with the least spread neutro-phils, showing the highest mobility (Fig. 6). The de-tachment at the cell tail and the extent of cellspreading now become the most likely obstructions toforward cell movement, and provides a plausible ex-planation for the observed variations in the neutrophilmobility.

In the presence of adsorbed plasma proteins, neu-trophil mobility increased on each of the three mate-rials, and was accompanied by concomitant decreasesin cell spreading area and cytoskeletal structures atcell–surface adhesion region. In addition, the densecytoskeletal ring around cell nuclei in neutrophils onglass and polyurethane surfaces suggest a well-formed cell cortex structure, which is essential for thegeneration of cell shape changes and extension oflamellipodia and filopodia and increased mobility.22,23

In the absence of adsorbed proteins, the interactionbetween a neutrophil and a material surface is likely to

occur through nonspecific intermolecular interactions,such as van der Waals, electrostatic, and hydrophobicinteraction. However, neutrophil surface receptorsmay still play an important role that may facilitate cellsignaling and promote the observed differences inneutrophil morphological responses.24,25 In the pres-ence of adsorbed proteins, specific interactions withneutrophil receptors were likely responsible for regu-lating cell responses.26

Material surface properties are known to affect thecomposition, conformation, and bioactivity of ad-sorbed proteins.27 This, in turn, will modulate thebinding of neutrophil receptors and the subsequencesignals that promote changes in the cell mobility andmorphology.28 It is the biological properties of theadsorbed protein layer that accounts for the relativecell speed of glass � polyurethane � OTS, and for thereversed order compared with materials in the ab-sence of adsorbed proteins. In general, hydrophobicbiomaterials lead to greater denaturation of adsorbedproteins, and disruption of cell–surface interactions.Consequently, the more hydrophilic materials (glass,polyurethane) should allow for least disruption ofprotein ligand–neutrophil receptor interactions andthe subsequent signal transduction events.

Assuming that neutrophil mobility is important inthe mechanism of bacteria phagocytosis in vivo, theresults have implications to implanted medical de-vices, because they will be coated with host plasmaproteins immediately following implantation.29 Theresults suggest that neutrophil mobility is retarded onhydrophobic materials. This may limit neutrophil ac-cess to adherent bacteria and increase the probabilityfor infection. Conversely, neutrophil functions may beenhanced through hydrophilic surface modification ofbiomaterials used in clinical applications.

CONCLUSIONS

The surface properties of materials directly influ-ence neutrophil–surface interactions, as determinedby cell morphological and cytoskeletal observations,and also affect cell mobility after stimulation by fMLP.In the absence of adsorbed plasma proteins, neutro-phil mobility increased with increasing material hy-drophobicity in the order of glass, polyurethane, andOTS. With plasma protein adsorption, cell mobilityincreased on each material, but followed the oppositematerial dependence, such that neutrophil mobilitydecreased with increasing material hydrophobicity.Surface mobility was inversely related to cell spread-ing, for stimulated cells.

The authors thank E. Colton, K. Yoshida, and J. Patel fortheir technical assistance.

BIOMATERIAL SURFACE-DEPENDENT NEUTROPHIL MOBILITY 619

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