Alteration of leukocyte motility on plasma-conditioned prosthetic biomaterial, ePTFE, via a...

Alteration of leukocyte motility on plasma-conditionedprosthetic biomaterial, ePTFE, via a flow-responsive celladhesion molecule, CD43

Rene S. Rosenson-Schloss, Charlie C. Chang, Alkis Constantinides, Prabhas V. MogheDepartment of Chemical and Biochemical Engineering, C230, Rutgers University, 98 Brett Road,Piscataway, New Jersey 08854-8058

Received 12 June 2001; accepted 14 June 2001

Abstract: The physiologic determinants of leukocyte mi-gration on vascular prosthetic biomaterials remain poorlyunderstood, despite their relevance to the control of peri-prosthetic infection. Using hemodynamic exposure of hu-man polymorphonuclear leukocytes adherent to expandedpolytetrafluoroethylene (ePTFE) in vitro, we investigated therole of fluid shear in regulating leukocyte migratory behav-ior on plasma-adsorbed, prosthetic vascular biomaterial.The presence of flow at a wall shear stress of 25 dyn/cm2

increased the degree of leukocyte displacement along theflow direction without altering the degree of overall cellattachment. Moreover, plasma-ePTFE elicited a lower over-all degree of displacement under flow in comparison withuntreated ePTFE. We further probed the molecular levelregulation of leukocyte migratory responses under flowthrough the immunocytochemical quantification of specificleukocyte adhesion molecules and determined that CD43, acell adhesion molecule, was upregulated via flow exposurefor leukocytes adherent to plasma-ePTFE, whereas basal lev-els of CD43 expression were not significantly altered on un-treated ePTFE. When flow-exposed, adherent leukocytes

were incubated in the presence of substrate immobilizedanti-CD43 immunoglobulin, the degree of cell displacementalong flow was found to be significantly enhanced onplasma-ePTFE. Quantification of the cell population redis-tribution under flow using a modified random motilitymodel, indicated that the incorporation of anti-CD43 onplasma-ePTFE led to a significant increase (243 ± 60%) in thecell dispersion coefficient, �D, whereas only a minimal in-crease (61 ± 30%) was detected on non-adsorbed ePTFE.Overall, our results suggest that flow exposure can inducethe migration of leukocytes adherent to prosthetic materialsin a substrate-dependent manner. An important implicationof our study is that, although biomaterials exposed toplasma intrinsically passivate leukocyte migration even un-der hemodynamic conditions, it may be possible to promotecell motility by targeting a specific, flow-responsive, adhe-sion molecule. © 2002 John Wiley & Sons, Inc. J BiomedMater Res 60: 8–19, 2002

Key words: leukocytes; physiologic shear; CD43; cell migra-tion; prosthetic biomaterial; ePTFE

INTRODUCTION

Vascular prosthetic materials, implanted during thereconstruction of damaged or occluded blood vessels,can be frequently compromised by the incidence ofbacterial infection.1,2 Because polymorphonuclear leu-kocytes (or neutrophils) serve as the key acute inflam-matory mediators,3 the control of neutrophil motility

on prosthetic biomaterial surfaces in particular may becritical to improve implant infection resistance.Whereas leukocyte motility behavior on normal vas-cular tissue has been extensively studied,4 much less isknown about the regulation of leukocyte migration onvascular prosthetic biomaterials, especially within theactive hemodynamic environment of the intravascularmilieu.

The intravasculature is characterized by the pres-ence of plasma proteins containing an excess of 100distinct moieties.5 After prosthetic material implanta-tion, circulating plasma proteins are rapidly adsorbedto biomaterial surfaces, thereby altering the interfacebetween cells and the biomaterial surface.6 We andothers have examined the effect of biomaterial ad-sorbed proteins on neutrophil morphology, migration,phagocytosis, and bacterial killing, and have deter-mined that such proteins can significantly modulatethe behavior of adherent neutrophils.7–12 In addition,the biomaterial surfaces can alter leukocyte adhesion

Correspondence to: P. V. Moghe; e-mail: [email protected] grant sponsor: Whitaker Foundation (biomedical

engineering research grant)Contract grant sponsor: Charles & Johanna Busch Bio-

medical Research Award (to P.V.M.)Contract grant sponsor: Rutgers/UMDNJ NIH Biotech-

nology Training Program (to C.C.C.)

© 2002 John Wiley & Sons, Inc.DOI 10.1002/jbm.1278

behavior by regulating the extent and nature of leu-kocyte adhesion molecules expressed,12–16 both in thepresence or absence of plasma proteins. Neutrophilmotility on vascular tissue is largely regulated by in-tegrin and selectin membrane adhesion proteins,which ensure efficient intercellular adhesive interac-tions.17 Leukocyte adhesion molecules are sensitivelyregulated by soluble inflammatory activators, and canbe further governed by variations in the substratephysicochemical properties and the biomechanical en-vironment.7,17,18 In our recent studies, we have iden-tified key integrin subunits mediating chemokineti-cally stimulated neutrophil migration on the pros-thetic material, expanded polytetrafluoroethylene(ePTFE).19 The possibility of substrate-specific alter-ation of neutrophil motility on prosthetic surfaces dueto physiologic hemodynamics, however, has not beenexamined.

The dynamic interaction between biomaterial matri-ces and normal blood flow includes not only serumprotein deposition but also accompanying shearstress, which operate along the biomaterial surfacesand cells adherent to these surfaces. Our previous re-sults demonstrated that physiologic shear can induceadherent macrophage activation typical of a stress re-sponse, and can alter both cellular morphology andfunction, underscoring the importance of shear stressinclusion in evaluating adherent leukocyte behavior.20

Whereas many have investigated the effect of physi-ologic fluid shear on attachment of circulating neutro-phils,17,21–24 the effect of shear on leukocytes adherentto prosthetic biomaterials has not been investigated.Prosthetic biomaterials fundamentally differ from na-tive vascular surfaces in that they can induce hyperad-hesive cell behavior via frustrated phagocytosis,which leads to high cell retention on the biomaterialeven during exposure to high rates of blood flow. Fur-ther, typical prosthetic biomaterials, even after plasmaprotein deposition, lack the ligands necessary to causetransient cell migratory phenomena such as cell roll-ing observed on endothelial cell layers. Thus, therestill remains unexplored the possibility of intrinsicmotility activation of neutrophils adherent to pros-thetic materials through flow exposure, and any li-gand regulation of motility on plasma-adsorbed ma-terials.

In this study, we assessed the effect of arterial levelshear stress on ePTFE-adherent neutrophils in aplasma protein environment. Our results indicate thatshear stress enhanced neutrophil polarity and inducedchemoattractant independent neutrophil migration onnon-adsorbed ePTFE surfaces; however, flow-inducedpolarity and migratory displacement were signifi-cantly lower on plasma-adsorbed ePTFE. We showthat flow exposure differentially modulates the ex-pression of an adhesion molecule, CD43, on plasma-adsorbed and untreated ePTFE, and that, in fact, this

molecule can be targeted to effectively promote theflow responsiveness of neutrophil migration on ePTFEsurfaces.

MATERIALS AND METHODS

Cell preparation

Human polymorphonuclear neutrophils (PMNs) wereisolated from peripheral blood as described previously.7

Briefly, 50 mL of blood was typically drawn from healthyvolunteer donors in accordance with the protocols estab-lished by the Rutgers University Institutional Review Board.The PMN fraction was isolated via a single step densitygradient centrifugation through neutrophil isolation media,NIM (Cardinal Associates, Santa Fe, NM) (2 volumes bloodwith 1 volume NIM) at 1380 rpm for 30 min at room tem-perature. The PMN-rich layer was aspirated, washed, andcentrifuged with Hank’s balanced salt solution (HBSS) with-out Ca2+ and Mg2+. To sediment the red blood cells, thePMN pellet was diluted in 0.15M NH4Cl lysing buffer andcentrifuged at 1000 rpm for 5 min. The PMN pellet was thenresuspended in HBSS at a concentration of 30 × 106 cells/mL(twice the desired final concentration). More than 95%PMNs were tested to be viable using trypan blue exclusion.

Before cell attachment, the biomaterial was adhered toglass slides with a thin coating of silicon sealant. For immu-nolocalization experiments, cells were directly attached tonon-adsorbed or plasma precoated 60-�m-porosity ePTFE(overnight 37°C incubation with normal human plasma)generously provided by W. L. Gore and Associates, Inc.(Flagstaff, AZ). After a 15 min, 37°C incubation, unattachedcells were gently removed, and ePTFE attached cells wereintroduced to the flow chamber. For cell migration studies,additional steps were undertaken during cell attachment todemarcate the boundary of seeded cells before the onset offlow (see Cell Migration Studies below; also see Fig. 1).

Flow studies

The flow system has been described in detail in our pre-vious study.20 Briefly, a temperature-controlled flow system

Figure 1. Schematic of the steps involved in cell chamberpreparation before leukocyte migration studies in the pres-ence of flow. For further details, refer to Materials and Meth-ods.

9LEUKOCYTE MOTILITY ON BIOMATERIALS UNDER FLOW

containing a parallel plate chamber was perfused with HBSSCa2+ and Mg2+ containing buffer at predetermined flowrates. The flow chamber was assembled with three compo-nents, a machined milled polycarbonate plate (Dr. JohnFrangos, University of California, San Diego, CA), a rectan-gular silicon gasket (0.03125 in.; Allstate Gasket and Pack-ing, Hicksville, NY), and a glass cover slide (38 × 75 mm;Fisher Scientific, Pittsburgh, PA). These components wereheld together by vacuum applied to the periphery of theslide to ensure consistent and uniform parallel plate assem-bly. The driving force for the flow loop was maintained bytwo reservoirs, situated one above the other with the parallelplate flow chamber positioned in between, as previouslydescribed.21

The cell-adherent slides were introduced into the chamberin an inverted manner to ensure that any detached cellswould be removed from the flow chamber by the fluidstream, minimizing the probability of random cell reattach-ment. Flow experiments were conducted at flow rates thatyielded an average shear stress value of 25 dynes/cm2. Thisshear level was selected because it is typical of physiologicarterial shear, and has been found to be optimal in modu-lating adherent leukocyte morphology and function.20 Qui-escent control conditions (0 dynes/cm2) were establishedeither by incubating inverted slides in a 37°C, 5% CO2 incu-bator, or by filling the chamber with buffer, bypassing flow,and incubating under static conditions in a 37°C water bath,which maintains constant temperature over the course of theexperiments. Both control conditions gave identical results.Flow experiments were conducted for 1 h. Adherent cellviability post flow exposure was confirmed by treatmentwith Calcein AM (Molecular Probes, Eugene, OR), whichgenerates a fluorescent cleavage product in metabolicallyactive cells.

Preparation of antibody-immobilized surfaces

Cells were attached as described above except that 20 �g/mL antibody was adsorbed overnight to ePTFE in the pres-ence or absence of plasma. As before, these antibody-basedmigration studies were performed at a fluid shear stressvalue of 25 dynes/cm2 because our previous studies attrib-uted maximum leukocyte polarity modulation, a predictorof cell motility, with this shear value.20 The stability ofePTFE-adsorbed antibody under flow was satisfactorilyverified by exposing ePTFE-adsorbed immunoglobulin(Ig)G conjugated with fluorescein isothiocyanate (FITC) to25 dyn/cm2 flow, and imaging before and after flow, underepifluorescence microscopy. Further, fluorescence intensityof ePTFE-adsorbed IgG-FITC was found to be comparable inthe presence or absence of plasma proteins.

Cell morphology studies

Slides containing neutrophils adherent to ePTFE were re-moved from the flow chamber, chemically fixed for 20 min

with 1% formalin, and permeabilized with 0.1% Triton-X for5 min at ambient temperature. Cells were then labeled withfluorescein conjugated phalloidin (Molecular Probes) di-luted 1:100, for 1 h at 37°C. Cells were washed three times inphosphate buffered saline (PBS) on a shaker platform, andimaged as described above. The major and minor axes ofcell-equivalent elliptic geometry were determined using Im-age-Pro image analysis software (Media Cybernetics, SilverSpring, MD). Cell morphologic polarity was computed asthe ratio of the length of major axis to the length of minoraxis.

Receptor immunolocalization analysis

Biomaterial-adherent neutrophils removed from flow orquiescent conditions were fixed for 20 min with 1% formalinat ambient temperature. Nonspecific binding was blockedby preincubation with 3% bovine serum albumin, 1% nor-mal goat serum in PBS overnight at 4°C. Cells were incu-bated with mouse anti-CD43, anti-CD44, anti-CD18, ormouse IgG1 (Pharmingen, San Diego, CA) for 30 min at 4°C,and then with goat anti-mouse IgG FITC, diluted 1:100(Boehringer-Mannheim, Indianapolis, IN) for 30 min at 4°C.Primary antibodies were used at a final concentration of 10�g/mL. Adherent cells were washed three times with PBSfor 10 min on a shaking platform after incubation with eachantibody. Specimens were excited at a peak wavelength of488 nm, under a Zeiss Axiovert 10 epifluorescence micro-scope, and fluorescent images (520-nm peak emission) wereacquired and analyzed using densitometry with Image-Proimage analysis software (Media Cybernetics). Specific fluo-rescence density was determined by subtracting the inten-sity of nonspecific fluorescence associated with IgG1 con-trols.

Cell migration studies

Neutrophils were seeded at very high density (6 × 106

cells/cm2) within selected regions of the biomaterial (seeFig. 1). Before attachment, the origin of the migration fieldwas defined by coating ePTFE with agarose and cutting a2.5-mm-wide well using a prefabricated plexiglas template.The agarose layer was gelled by cooling a 2% w/v solutionof Indubiose A37 agarose (Biosepra, Marlborough, MA) in a60°C water bath, and combined with an equal volume ofgelatin solution (bovine skin gelatin; Sigma, St. Louis, MO),to achieve final concentrations of 1% w/v agarose and 0.5%w/v gelatin. After cell attachment, the unbound cells weregently removed and then the agarose layer was removed.Experiments were carefully designed to preclude the possi-bility of cell dispersal by physical manipulation. Finally, theePTFE slides seeded with neutrophils were exposed to flowor control conditions. After 1 h of incubation, the slides wereremoved from the flow chamber, cells were chemically fixedand permeabilized as described above, and the nuclei werelabeled with 2 �M ethidium bromide (Molecular Probes) to

10 ROSENSON-SCHLOSS ET AL.

visualize cells. Two hundred-micrometer-wide sectionswere imaged in triplicate across 50-�m-long increments andthe number of cells was assessed in each section via thresh-olding and densitometry, using Image Pro software (MediaCybernetics) on a Dell Dimension XPS Pro200n PC worksta-tion. The constancy of cell concentration at the boundary, Co,was experimentally verified under various conditions; thetypical range was 24–29 cells per 104 �m2. Variations inexperimentally averaged values of Co values between qui-escent and flow conditions were less than 5% for bothplasma-ePTFE and untreated ePTFE, and these variationswere not statistically significant even after 1 h of incubation(p > 0.05). Differences between initial and final (t = 1 h)values of Co were also minor (∼5–8%), and, as such, experi-mentally averaged values of Co at t = 1 h were used in themodel calculations described below.

The possibility of nonspecific displacement of weakly ad-herent cells immediately after flow exposure (2 min) wasassessed for all substrate conditions (bare/plasma/anti-CD43 adsorbed/IgG adsorbed). Cell distribution profileswere found to be unchanged from the preflow profiles, thusruling out any nonspecific reattachment of cells downstreamunder flow; further, the number of initially adherent cellswas also unchanged, pointing to the hyperadherent re-sponse of neutrophils to ePTFE.

Characterization of cell migration under flow

Cell motility on ePTFE was characterized in terms ofquantitative parameters describing population-level redis-tribution upon flow exposure. After the flow incubation wascompleted, the cell number, C, corresponding to spatial co-ordinate along flow direction, z, was computed by measur-ing the number of adherent cells in the 50-�m sections cen-tered around z, and normalizing cell number to Co, the num-ber of adherent cells in the boundary region. The spatialchange in normalized cell concentration, C, was fitted to ageneric model for random and biased migration to computethe migration coefficient.

It is not straightforward to experimentally discern the ran-dom and directional modes of migration under the highlevels of imposed flow rates, because the movement of evenrandomly migrating cells can be directionally constrained.We attempted to quantify the migratory response using twoseparate approaches, as elaborated in the Appendix. Onlythe second approach (Approach 2 detailed in the Appendix)could be used to compute migratory parameters with satis-factory statistical confidence, and, as such, this approachwas used to yield estimates for pseudo-random migratorycoefficient, �D. This approach entails a nonlinear regressionof the cell redistribution data using Equation (E2b) in theAppendix. In each case, the migration model was fitted to allthe experimental data using the nonlinear regression pro-gram (NLR) developed by A. Constantinides and N. Most-oufi (Numerical Methods for Chemical Engineers with MATLABApplications, Prentice Hall, 1999). This program uses theMarquardt method of nonlinear regression and performs acomplete statistical analysis on the regressed results. A totalof six sets of data, each set consisting of four repeated ex-periments, were analyzed.

Statistical analysis

Statistical analyses of cell polarity data were performedusing unpaired t test assuming unequal variances. The errorbars indicate the standard error around the mean data val-ues obtained from at least three independent experiments (n� 3). Regression data for cell migration under flow wasanalyzed using a number of statistical measures: (a) the val-ues of the regressed parameters, with the 95% confidenceintervals; (b) testing of the null hypothesis that the value ofthe parameter is equal to zero, using a two-sided t test; (c)standard deviation of the parameters and the number ofdegrees of freedom; (d) randomness of the distribution ofpositive and negative residuals, as determined by the Runstest; (e) sum-of-squared residuals; and (f) analysis of vari-ance.

RESULTS

Effect of physiologic fluid shear on leukocytemorphology and migration on ePTFE

The morphologic changes of ePTFE-adherent neu-trophils after exposure to 0 or 25 dynes/cm2 arteriallevel shear were compared. The results, summarizedin Figure 2(a,b), indicate that in the absence of shearstress, neutrophil 2-D polarity was lower on plasma-coated ePTFE surfaces than on untreated ePTFE. How-ever, arterial level shear significantly increased cellpolarity on both ePTFE and plasma-pretreated ePTFEsurfaces. However, whereas polarity values averagedalmost 2.0 on untreated ePTFE surfaces, these valueswere significantly lower on plasma-coated surfaces,even after fluid flow exposure.

The spatial displacement of neutrophils adherent tothese ePTFE surfaces and exposed to 25 dynes/cm2

shear was assessed. Figure 3(a) indicates that themaximum displacement on non-adsorbed ePTFE wasabout 300 �m greater than that observed for neutro-phils studied under quiescent control conditions. Al-though neutrophils attached to plasma precoatedePTFE surfaces also exhibited detectable levels of dis-placement in the presence of fluid shear [Fig. 3(b)], themaximal migrated distances were significantly loweron plasma-coated surfaces (0.029 ± 0.003 cm; n = threeexperiments) than those observed under correspond-ing flow conditions on non-adsorbed ePTFE surfaces(0.053 ± 0.004 cm; n = 3). Plasma pretreated ePTFEthus inhibited both shear induced neutrophil polariza-tion and motility.

Leukocyte adhesion receptor responsivenessto flow

We examined the effect of flow exposure on thevariations in the membrane expression of three neu-


Figure 2. (a) High magnification (original magnification ×100) fluorescent micrographs of fluorescein phalloidin stainedhuman neutrophils adherent to non-adsorbed ePTFE under quiescent (A) or 25 dyn/cm2 flow (B), and of neutrophils adherentto plasma-adsorbed ePTFE, under quiescent (C) or 25 dyn/cm2 flow (D). The magnification bar = 10 �m. Neutrophils wereexposed to flow or quiescent conditions for 1 h. (b) Substrate-specific variation in neutrophil morphologic polarity on ePTFE,under quiescent and flow conditions. PMN polarity reflects the ratio of lengths of the major and minor axes. Data representthe average of three independent experiments, involving 25 cells each.


trophil adhesion receptors, CD43, CD44, and CD18.The substrate-specific expression of these surface pro-teins within neutrophil populations is presented inFigure 4. Whereas there was a basal expression ofCD43 on neutrophils attached to both non-adsorbedand plasma-ePTFE surfaces in the absence of flow, inthe presence of 25 dynes/cm2 shear stress, a signifi-cant elevation in CD43 expression was induced onplasma precoated but not non-adsorbed surfaces. Incontrast, neither CD44, another cell surface adhesionprotein, nor CD18, the � chain of �2 integrins, wassignificantly elevated by flow on cells attached toplasma-coated or non-adsorbed ePTFE surfaces.

Role of CD43 in flow-inductiveleukocyte migration

The possible mediation of cell migration by CD43molecule was assessed by examining the effect of sub-

strate adsorbed anti-CD43 antibody on flow-inducedneutrophil migration on ePTFE. Low magnificationmicrographs of differential levels of population-levelcell displacement along the flow direction appear inFigure 5(A–D). On anti-CD43 immobilized, plasma-adsorbed ePTFE surfaces, the extent of neutrophil mi-gration was found to be increased when comparedwith control IgG immunoglobulin [see data points inFig. 6(a)]. Furthermore, anti-CD43 also increased neu-trophil migration on non-adsorbed ePTFE surfaces, al-though the degree of change was detectably lower[data points appear in Fig. 6(b)]. In contrast, anti-CD44-coated ePTFE (positive controls for adhesion)did not elicit statistically significant differences in celldensity distribution upon flow exposure (data notshown).

Characterization of flow-inductiveleukocyte migration

Flow-mediated cell redistribution was quantified bycalculating random motility coefficient values, ex-

Figure 3. Representative cell density profiles for neutro-phils exhibiting migration outward from a cell well on (a)non-adsorbed ePTFE, or (b) plasma-adsorbed ePTFE. Neu-trophils were seeded in a cell well to the left and exposed toflow or quiescent conditions for 1 h. For each experiment,values of cell density (C) were normalized to those observedat the cell well boundary (Co). The curves are representativeof data from three independent experiments.

Figure 4. Frequency distribution of the immunofluores-cence intensity of specific membrane receptors (CD43, CD44,and CD18) expressed on ePTFE-adherent neutrophils thatwere exposed to flow (25 dyn/cm2) or quiescent conditionsfor 1 h. The receptor-specific fluorescence intensity was cor-rected by subtracting the background fluorescence for thecorresponding isotype antibody. Data shown are represen-tative of four independent experiments.


pressed as �D. Our mathematical approach is summa-rized in the Appendix. Regression analyses indicatedthat the cell distribution profiles were satisfactorilydescribed using a pseudo-random dispersion/diffusion model [see model fits in Fig. 6(a,b); data inTable II of the Appendix]. The percent change in flow-induced population-averaged migration coefficient onanti-CD43-treated, non-adsorbed, and plasma-coated

surfaces were normalized to their respective flow con-trols and are presented in Figure 7. Levels of randommotility (�D) on plasma-ePTFE were found to be sig-nificantly elevated by the presence of CD43 antibodyimmobilized surfaces. The relative increase on non-adsorbed ePTFE surfaces was found to be less pro-nounced (p < 0.1). It should be noted that on ePTFEadsorbed with anti-CD44 (CD antigen control), �D val-ues on non-adsorbed ePTFE and plasma precoatedePTFE were not statistically distinguishable from thevalues on control IgG-adsorbed surfaces (data notgraphed).

DISCUSSION

The activation of neutrophils on prosthetic bioma-terial surfaces has been traditionally attributed to the

Figure 5. Low magnification confocal laser scanned micro-graphs of population level PMN migration on ePTFE, afterexposure to 25 dyn/cm2 flow for 1 h and with specific sur-face pretreatment: (A) ePTFE with anti-CD43 antibody, (B)ePTFE with IgG controls, (C) plasma-ePTFE with anti-CD43antibody, and (D) plasma-ePTFE with IgG controls. (Bar =100 �m; flow direction is to the right and parallel to the bar.)

Figure 6. Representative cell density profiles for neutro-phils exhibiting migration outward from a cell well on (a)plasma-adsorbed ePTFE, or (b) non-adsorbed ePTFE, bothimmobilized with 20 �g/mL anti-CD43 antibody. The anti-body-specific control substrates were immobilized with 20�g/mL IgG. Neutrophils were seeded in a cell well to theleft and exposed to flow or quiescent conditions for 1 h.Values of cell density (C) were normalized to those observedat the cell well boundary (Co). The profiles are representativeof data from four independent experiments.


physicochemical properties of the material, includingthe material texture, chemistry,25 surface energetics,26

and to the exogenous chemical stimulation by solublechemoattractants.7,18 The role of mechanical fluidshear in effecting activation events in neutrophils hy-peradherent to prosthetic materials has not been welldocumented. The current investigation examined theeffect of physiologic fluid shear on the migratory re-sponses of neutrophils adherent to a widely implantedprosthetic material, ePTFE.27 The results of our studiessuggest that arterial level fluid shear in vitro can in-duce neutrophil polarization and chemoattractant in-dependent migration on ePTFE, and that the presenceof adsorbed plasma protein on ePTFE can limit theflow-induced cellular migration. We have also ob-served that flow induction of neutrophil migration onePTFE may be tightly coordinated by specific adhe-sion receptors such as CD43, whose role may bemodulated both via the degree of fluid shear as well asthe protein environment.

CD43 (or leukosialin) is a heavily sialated trans-membrane glycoprotein,28 and is widely regarded asan anti-spreading molecule, attributed in part to its netnegative charge and its extraordinarily long two-dimensional structure which extends 45 nm.29 Recentstudies have also implicated CD43 proteolytic cleav-ing and shedding as a prerequisite for cell spreading.29

Albumin, a major component protein of blood plasma,is known to interfere with this shedding event.11 Thesereported findings are consistent with our studies, be-cause flow-activated neutrophils on plasma-coated,

but not non-adsorbed, ePTFE surfaces demonstratedconsistent enhancement in CD43 expression, a novelobservation for this important neutrophil adhesionmolecule. Thus, although flow-induced CD43 upregu-lation may occur on both untreated and plasma-coated surfaces, in the absence of plasma components,the CD43 cleavage on untreated ePTFE surfaces mayhave resulted in the lower CD43 expression detectedin our studies. It is intriguing that, under quiescentconditions, neutrophil motility levels were compa-rable on non-adsorbed and plasma-ePTFE; however,under flow, migratory activation on plasma-ePTFEwas relatively inhibited. One significant factor under-lying these phenomena may be the particular upregu-lation of CD43 molecular expression on flow-exposedplasma-ePTFE adherent neutrophils. Indeed, onplasma, the CD43 molecule may be “stabilized” viacomplexing with albumin from plasma,11 and couldpotentially inhibit the rate of cell movement throughsteric blockage and electrostatic repulsion at thelargely negative ePTFE surface (W. L. Gore and Asso-ciates, personal communication). Our findings sup-port this premise, because flow-exposed neutrophilson plasma-adsorbed ePTFE exhibited enhanced mi-gration specifically in the presence of anti-CD43, butnot anti-CD44 immobilized ePTFE surfaces. Thus, re-versal of CD43 molecular inhibition may allow theengagement of native motility pathways to proceedrelatively unimpeded, for example, that of �2 integrin-mediated migration, which we have previouslyshown to regulate chemosensory leukocyte migrationon ePTFE.19

It is noteworthy that neutrophils exhibit high levelsof adhesion to the prosthetic substrate,18 regardless ofthe nature of substrate pretreatment used here. Basedon preliminary studies, even after flow exposure, wedid not observe a significant variation in the totalnumber of attached cells on these substrates (confirm-ing neutrophil hyperadherence to ePTFE). Moreover,the surface incorporation of anti-CD43 did not appearto detectably alter the net extent of cell attachmentunder flow conditions. The adhesion-modulating roleof CD43 pertinent to cell migration is still unclear,although we can speculate that the charged and albu-min-complexing nature of CD43 may be neutralizedby the incorporation of anti-CD43 antibody, thus al-tering the differential kinetics of local cell-substrateattachment and detachment. We will test the validityof this hypothesis in future studies using ePTFEtreated with albumin-deficient plasma.

The role of CD43 on cell morphologic polarizationand orientation appears tenuous, especially underconditions superimposed by high shear flow condi-tions. Thus, although anti-CD43-mediated receptorcrosslinking could theoretically cause cytoskeletal re-organization and cell shape changes,30–34 we did notdetect any change in cell polarity beyond the levels

Figure 7. Normalized anti-CD43-mediated modulation ofpopulation-averaged leukocyte random migration coeffi-cient, �D, in response to 25 dyn/cm2 fluid shear stress and inthe presence of immobilized immunoglobulin (20 �g/mL)on non-adsorbed or plasma-adsorbed ePTFE surfaces. Thedifferential effect of anti-CD43 adsorption on plasma com-pared with non-adsorbed ePTFE surfaces is expressed as(�CD43 − ⟨�Ig⟩)/⟨�IgG − free

⟩) × 100. The error bars reflect thestandard error around the mean values of �CD43 values fromfour independent experiments.


induced at 25 dyn/cm2 flow on both plasma-treatedand non-adsorbed ePTFE. We believe that increase inanti-CD43-mediated migration is not likely to be ac-companied by “further” changes in the basal morphol-ogy specific to the substrate under high levels of flow,although polarity can affect leukocyte motility inmany systems35 and is correlated with neutrophil mi-gration on non-antibody adsorbed ePTFE surfaces inour studies. Similarly, increased downstream dis-placement under flow on anti-CD43 plasma-ePTFEdid not appear to correlate with any increase in the cellorientation bias along flow (data not shown). This isnot unexpected because of two intrinsic orientingcues: the ePTFE substrate, comprising 5–8-�m-widemicrochanneled fibers, which structurally limit therange of possible cell reorientation along the flow di-rection; and high shear flow. A recent study on neu-trophil migration on more weakly adhesive, endothe-lial cell layers, reported that integrin �v�3 receptorinhibition randomized flow-induced cell orientation,without altering the cell migration velocity.36 In con-trast, our work suggests that, under conditions of geo-metrically imposed orientation along 25 dyn/cm2

flow, the substrate incorporation of the anti-CD43 an-tibody does promote neutrophil displacement rate. Itshould be noted that, whereas the direct visualizationof the migration behavior of unlabeled cells on opaqueePTFE disks under flow is still not possible, our pre-liminary studies establish that even flow “pre-exposure” can activate cell migration in a substrate-specific manner.

Although our data for cell redistribution under flowunequivocally shows anti-CD43-induced alteration ofcell displacement on plasma-ePTFE, the quantitationof this observation, utilizing approaches similar toTranquillo et al.,37 for chemoattractant-stimulatedneutrophil migration, was not straightforward. Thiswas attributed, in part, to restrictions of direct imagingon opaque materials under flow. We have, therefore,modeled the population cell redistribution in terms ofa single, lumped motility parameter, indicative of apseudo-random dispersive/diffusive process. Analo-gous lumped diffusion models have been used for cellmigration in the presence of externally orientedfields.38 Our current modeling approach is a first ap-proximation, and will need to be refined in futurestudies with both convective and diffusive componentfits to data obtained for cell distribution profiles ob-tained over a wider range of temporal conditions.However, our regression analyses of cell populationredistribution after flow supported the hypothesis thatchanges in the rate of cell motility (measured after 1 hof flow exposure), likely resulted not from nonspecificconvective displacement arising from cell detachment,but rather from an active migration process on a mi-crogeometry under a superimposed flow field.

Regardless of the underlying mechanism, we have

demonstrated that flow can directly engender motil-ity-associated redistribution of otherwise hyperadher-ent neutrophils on prosthetic biomaterial surfaces,even in the absence of an exogenous chemoattractant.The CD43 molecule may modulate flow-induced neu-trophil motility by interfering with plasma protein in-hibitory effects. Finally, CD43 crosslinked surfacesmay provide a novel flow-responsive paradigm toregulate neutrophil migration rates under physiologicconditions, and may potentially serve as a basis todevelop improved infection-resistant prosthetic, vas-cular materials.

APPENDIX

Parametric analysis of cell migration under flow

Two different approaches were considered duringthe estimation of cell migratory parameters on the bio-material substrates under flow conditions. We presentthese approaches herein sequentially for complete-ness.

Approach 1 (Regression of strictly random andbiased migratory components attemptedsimultaneously)

Here, cell distribution profiles under flow were thenanalyzed using a diffusion-convection model asshown in Equation (E1). The overall time-variant cellconcentration was formulated in terms of a directionaldisplacement component comprising the directionalmigratory velocity (veff), which was defined based ona reference migratory rate, �D

�c�t

= �D

�2c

�z2 − veff

�c�z

[E1]

with initial and boundary conditions as follows:

t = 0, all z > 0, c = 0t > 0, z = 0, c = c0

z = �. c = 0

Equation [E1] was integrated using Laplace transfor-mation to yield the following solution [E2]:

cc0

= exp�veffz2�D

��12

exp�−zveff

2�D�

erfc�� z2

4�Dt−�veff

2t4�D

� +12

exp�zveff

2�D�

erfc�� z2

4�Dt+�veff

2t4�D

��


Experimental values of cell density at various z coor-dinates were fitted to [E4] using nonlinear least-squares regression in MATLAB 5.3 (Release 11.1; TheMath Works, Inc., Natick, MA), to yield estimates forboth �D and veff for non-adsorbed and plasma-treatedePTFE (Table I). The null hypotheses were tested onall the values of the parameters using the t test. Thevalues of �D for all six sets of data were found to besignificantly different than zero, i.e., the null hypoth-esis was rejected. The distribution of positive andnegative residuals was random for three of the six sets(not shown for brevity). However, the null hypothesisfor veff could not be rejected (H: veff = 0) for four of sixsets of data. From this statistical analysis, we infer thatthe contribution of veff was likely negligible under theexperimental conditions used.

Approach 2 (Single lumped, pseudo-randomcomponent regressed)

Based on the results of the previous case, in thesubsequent analysis we set the value of veff to zero andfitted the modified model to the data (Table II). As alumped, single parameter, �D thus reflects a pseudo-random cell migration coefficient, and can equiva-

lently be derived from the classical inverse error func-tion profile of concentration with respect to space andtime, shown in Equation (E2b).18

erfc − 1� CC0� =

z

�4�Dt[E2b]

All results discussed in the study reflect �D valuesobtained via Approach 2 above. The values of �D forall six sets of data were found to be significantly dif-ferent than zero, i.e., the null hypothesis was rejected.The distribution of positive and negative residualswas random for four of the six sets, and the values ofthe sum-of-squared residuals were low. The resultsfrom the analysis of variance were satisfactory, i.e., theratios of s2

fit/s2expwere smaller than the F0.95(vfit,vexp)

statistic in all experiments. This means that the com-ponent of variance due to the lack of fit is statisticallysmall compared with the variance of the experimentalerror, indicating that the model adequately fit thedata. The statistical results of this case were more sat-isfactory than the first approach; therefore, resultsfrom this approach were incorporated in our finalanalysis and conclusions.

This study was supported by a Whitaker Foundation Bio-medical Engineering Research Grant and a Charles & Jo-

TABLE IValues of µD and veff were Estimated by Regression

Experiment�D ± 95% CI

(cm2/s)Null hypothesis

H0: �D = 0vaff ± 95% CI

(cm/s)Null Hypothesis

H0: veff = 0

A: Plasma-ePTFE flow condition (with CD43) 15.59e-8 ± 7.22e-8 Reject −1.66e-7 ± 35.83e-7 AcceptB: Plasma-ePTFE flow condition (with IgG) 2.73e-8 ± 1.74e-8 Reject 25.19e-7 ± 13.38e-7 RejectC: Plasma-ePTFE flow without pretreatment 1.92e-8 ± 0.91e-8 Reject 17.53e-7 ± 8.95e-7 RejectD: ePTFE flow condition (with CD43) 15.15e-8 ± 4.65e-8 Reject −28.11e-7 ± 48.49e-7 AcceptE: ePTFE flow condition (with IgG) 7.50e-8 ± 5.00e-8 Reject 5.86e-7 ± 30.69e-7 AcceptF: ePTFE flow without pretreatment 9.61e-8 ± 3.0e-8 Reject −24.3e-7 ± 33.09e-7 Accept

CI = confidence interval.

TABLE IIThe Value of veff Was Set to Zero and the Lumped µD Was Evaluated by Regression

Experiment�D ± 95% CI

(cm2/s)Null Hypothesis

H0: �D = 0StandardDeviation

Sum-of-SquaredResiduals s2

fit/s2exp F0.95(vfu,vexp)

A: Plasma-ePTFE flowcondition (with CD43) 15.24e-8 ± 1.67e-8 Reject 0.83e-8 0.2909 0.447 1.95

B: Plasma-ePTFE flowcondition (with IgG) 6.20e-8 ± 1.24e-8 Reject 0.61e-8 0.3269 1.143 2.36

C: Plasma-ePTFE flowwithout pretreatment 3.77e-8 ± 0.56e-8 Reject 0.27e-8 0.1379 2.200 2.42

D: ePTFE flow condition(with CD43) 13.64e-8 ± 1.77e-8 Reject 0.88e-8 0.4662 0.895 1.88

E: ePTFE flow condition(with IgG) 8.46e-8 ± 1.32e-8 Reject 0.65e-8 0.3863 0.935 2.05

F: ePTFE flow withoutpretreatment 8.84e-8 ± 0.94e-8 Reject 0.46e-8 0.1871 0.656 2.04

CI = confidence interval.


hanna Busch Biomedical Research Award to Prabhas Mo-ghe. Charlie Chang was partially supported by the Rutgers/UMDNJ NIH Biotechnology Training Program. Thegenerous donation of ePTFE from W. L. Gore & Associates,Inc. is sincerely appreciated.

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