Biomaterial-induced alterations of neutrophil superoxide production S. S. Kaplan,'.* R. E. Basford; E. Mora? M. H. Jeong? and R.L. Simmons3 Departments of 'Pathology, 'Molecular Genetics and Biochemistry, and 3Surgery, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213-2582 Because periprosthetic infection remains stimulation via protein kinase C with a vexing problem for patients receiving phorbol myristate acetate, however, was implanted devices, we evaluated the ef- not consistently observed. The cells evalufect of several materials on neutrophil free ated for 02-release during continuous asradical production. Human peripheral sociation with the biomaterials showed blood neutrophils were incubated with enhanced metabolic activity during short several sterile, lipopolysaccharide (LPS)- periods of association (especially with f r e e biomaterials u s e d i n surgically polyurethane and woven dacron). Alimplantable prosthetic devices: polyure- though 02-release by neutrophils in asthane, woven dacron, and Velcro. Free sociation with these materials decreased radical formation as the superoxide (02-)with longer periods of incubation, it was anion was evaluated by cytochrome c re- not obliterated. These studies, therefore, duction in neutrophils that were exposed show that several commonly used bioto the materials and then removed and in materials activate neutrophils soon after neutrophils allowed to remain in associa- exposure and that this activated state dition with the materials. Neutrophils ex- minishes with prolonged exposure but posed to polyurethane or woven dacron nevertheless remains measurable. The for 30 or 60 min and then removed con- diminishing level of activity with prosistently exhibited an enhanced release longed exposure, however, suggests that of 02-after simulation via receptor en- ultimately a depletion of reactivity may gagement with formyl methionyl-leucyl- occur and may result in increased suscepphenylalanine. Enhanced reactivity to tibility to periprosthetic infection.

INTRODUCTION

Artificial materials (biomaterials) have been used as effective substitutes for diseased organs and tissues for more than 30 years. Both permanent implants, such as artificial joints'-3 and vascular prostheses,4-' and temporary implants, such as the artificial heart: and the left ventricular assist device,'" have been successfully employed. Bacterial infection in the periprosthetic space, however, has remained a vexing source of morbidity and The pathogenesis of these infections is thought to be due not only to properties of the biomaterial that increase microbial adherence but also to an impaired host defense in the region of the implant.'",16 *To whom correspondence should be addressed.

Journal of Biomedical Materials Research, Vol. 26, 1039-1051 (1992) ccc 0021-9304/92/0SlO39-13$4.00 0 1992 John Wiley & Sons, Inc.

KAPLAN ET AL.

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Neutrophils are crucial effector cells in host defense against microbial invaders. Their effective functioning requires an ability to respond to infection by translocating to the site of contamination and then by ingesting and killing the microorganisms. Killing, in turn, requires the phagocytosis-associated metabolic activation of the neutrophil, leading to intraphagosomal production of activated oxygen species such as the superoxide anion (02-) together with the release into the phagocytic vacuole of lysosomal constituents such as myeloper~xidase.'~ It is well known that substances other than microorganisms may activate ne~trophils.'~"~ Not only do soluble stimuli (formyl peptides, C5a, LTB4) cause metabolic activation and secretion of granule constituents, but nonphagocytosable surfaces do as well.20Such metabolic activation and secretion itself can result in inflammatory changes and tissue injury or degeneration.21It is likely that implantable biomaterials also provide surfaces that can stimulate neutrophils.22Such stimulation not only could result in tissue damage but could also result in a depleted neutrophil poorly able to respond to an infectious challenge. Zimmerli studied biomaterial-neutrophil interactions and showed that the presence of a nonphagocytosable foreign body (Teflon) in vivo induced a neutrophil defect after 14 days that was manifested by poor phagocytosis, poor bactericidal activity, especially for catalase-positive organisms, poor metabolic activity, and diminished granule content. These defects could be reproduced in vitro by the presence of large quantities of finely divided Teflon." Marchant et al., using polyurethane implants in rats, found that neutrophils and macrophages became associated with the implanted material and that their phagocytic capacity decreased with time.23Prior investigations, however, have not evaluated the early effects of the biomaterial/neutrophil interaction and did not address the specific cellular defect that may cause decreased phagocytic capacity. To explore this early interaction between biomaterial and neutrophil, experiments were designed to evaluate in vitro the effect of incubating neutrophils with a group of biomaterials presently used for the construction of implants in clinical practice. Our results demonstrated that all the materials tested induce transient increases in oxidative metabolism by neutrophils and that after longer periods of incubation the remaining neutrophil population exhibited decreasing responsiveness to stimulators of activation.

EXPERIMENTAL

Cells and reagents Blood was obtained from normal human donors, chiefly medical students, coworkers, and employees. Donors were questioned about their health status and medication usage and were not used during periods of illness or medication use. Venus blood (60-120 mL) was obtained from each donor and the blood was anticoagulated with heparin at 6 U/mL. All subjects enrolled in

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this research responded to an informed consent that had been approved by the Institutional Committee on Human Research and found acceptable (latest IRB approval date 9/12/90). Neutrophils were isolated in lipopolysaccharide (LPS)-free medium by density gradient centrifugation by a modification of Boyum’s techniquez4as previously de~cribed.’~ Briefly, the blood was layered over Ficoll Hypaque (Sigma) and centrifuged at 800X g for 20 min. The neutrophil-rich lowest layer was diluted with an equal volume of buffer described below plus 2 mL 6% dextran to sediment the red blood cells (RBCs). The neutrophil-rich supernatant was removed, centrifuged, washed with buffer, and resuspended in buffer. Each neutrophil donor provided cells for a single day’s experiment. The preparations were uniformly greater than 90% neutrophils as determined by microscopic examination of a cytospin preparation stained with Diff Quick (Baxter Healthcare Corp., Miami, FL). Viability was determined by trypan blue dye exclusion and was greater than 95% in all cases. The neutrophils were counted with a Coulter ZBI (Coulter Electronics, Hialiah, FL) automated cell counter and the cells were suspended in Krebs ringer phosphate buffer pH 7.4 (16 mM Na2HP04,123 mM NaC1, 0.5 mM CaCl,, 0.5 mM MgS04, 5 mM KC1, and 4.4 mM glucose) (KRPG) at a concentration of 1-2 X 107/mL. In some experiments, the buffer contained 10% heat-inactivated calf serum (GIBCO). Stock solutions of the peptide formyl methionyl-leucylphenylalanine(fMLP) and of phorbol myristate acetate (PMA) (Sigma Chemical Co., St. Louis, MO) were prepared in DMSO and stored at -20°C. These activators were further diluted with KRPG for daily use.24 Biomaterials

The biomaterials were generous gifts and include: segmented polyether polyurethane (Symbion, Inc., Salt Lake City, UT), Velcro felt (Three Rivers Supply Co., W. Homestead, PA), woven dacron Cat. No. 174426 (Meadox Medicals, Inc., Oakland, NJ), and polyethylene NIH NHLBI-DTB reference material (Abiomed, Inc., Danvers MA). These were cut into 13 X 13 mm squares, sterilized with ethylene oxide, and degassed for 1 d. The squares were fit into the bottom of the wells in Corning 24-well tissue culture plates. In the studies where the polystyrene of the tissue culture dishes served as a control, 600 pL calf serum was added to the wells for 90 min at 37°C. The remaining serum was removed and the wells were washed once with 1 mL sterile saline and allowed to dry. LPS decontamination

All materials were processed to remove LPS or were purchased as LPS free. Glassware was baked at 200°C for 4 h and only LPS-free water was used. Pipet tips were soaked with 1%E-toxate solution (Sigma) overnight, washed until shown to be LPS negative, then autoclaved. Velcro was cut into 26 X 91 mm pieces, placed in H,O containing 10 ng/mL polymyxin B, and

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KAPLAN ET AL.

stirred for 4 h. The water was discarded and replaced with fresh HzO six times. All cut biomaterials were placed into glass Petri dishes and autoclaved for 15 min at 120°C. When cool, 10 mL water was added, swirled to make contact with the biomaterials, and allowed to sit overnight. The water was tested for the presence of endotoxin according to the Sigma No. 210 bulletin. Materials or solutions containing LPS were not used. This was necessary to ensure that any changes in neutrophil activity were biomaterial, rather than LPS induced.

Metabolic studies Two kinds of metabolic studies were performed (Fig. 1 flow sheet): (1) In the first type, the neutrophils in KRPG (1mL total volume containing 1 X lo7 cells) were placed on the biomaterials that had been fit into the bottom of the wells of 24-well culture dishes. These neutrophils were removed from the presence of the biomaterials after the following lengths of association time: 0, 30, and 60 min. Neutrophils in contact with the biomaterials for 30 and 60 min were incubated at 37°C upon a Koala-Ty slow variable rotating shaker (Accurate Chemical and Scientific Corp., Westbury, NY) at 90 oscillations/min. Only the cells remaining in suspension or readily resuspended with gentle agitation were removed. Viability of the neutrophils was reassessed by trypan blue dye exclusion and was determined to be >90% and the cells were recounted with the Coulter ZBI automated counter. The neutrophils were then added to the wells of 96-well Falcon flat-bottomed microtiter plates (Fisher Scientific, Pittsburgh, PA). Quadruplicate samples containing 3-4 X lo5 neutrophils for each condition were pipetted into the wells. Superoxide dismutase (SOD) 50 pg/mL (3000 U/mg) was added to sample 4 and cytochrome c, 200 pm, was added to all the wells. Three conditions were assessed: (i) absence of an additional stimulus, (ii) additional stimulation with fMLP at M, or (3) additional stimulation with PMA 1 pg/mL. Neutrophil release of superoxide was evaluated by measuring the superoxide dismutase inhibitable reduction of cytochrome c at 550 nm using a Molecular Devices Corp. (Menlo Park, CA) microtiter plate reader."j Determinations of the amount of cytochrome c reduced were carried out every minute for 10 min. Superoxide was calculated using an extinction coefficient of 0.0131 taking into account a light path of 0.6 cm and a volume of 200 pL. (2) The second type of experiment was designed to evaluate neutrophils superoxide release during the period of contact with the biomaterials. These experiments were carried out in the 24-well culture dishes and were termed in situ experiments since the neutrophils were not removed from the presence of the materials. In these experiments, serum-coated polystyrene material and polyethylene served as nonactivating negative controls. Nonactivating conditions on polystyrene also could be obtained by including 10% calf serum in the KRPG. Several experiments, including all the woven dacron studies, were conducted in this manner. We demonstrated that the presence of 10%

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NEUTROPHILS Isolate, Quantitate, Assess Viability

L Type A Studies Post Contact Activity 1.

24 Well Culture Dishes

containing

biomaterials

+ Neutrophils,

5 5 x 10 / m l

in KRPG

Type B Studies In Situ Activity

I

24 Well Culture Dishes containing biomaterials

I

+ Neutrophils. 5 x 1 0 5 / m 1 in KRPG - 10% calf serum

INCUBATE 0, 30, or 60 min.

INCUBATE 10, 30, or 120 mio

31 O C

37oc

I

2.

Remove Neutrophils from Biomaterials. Count and assess viability

Add cytochrome c f Activators fS[D to Neutrophils in B-1

INCUBATE 30 min., 37 0 C

3.

Transfer Neutrophils to 96 well culture dishes Add cytochrome c f Activators

3.

*-

as it is occurring

I

Chill culture dishes to 4 OC CENTRIFUGE Transfer cell-free supernatant to 96 well dishes

4.

I

Measure 0; r e l e a s e that had occurred in the 24 well dishes (B-2)

Figure 1. Flow diagram describing incubation of neutrophils with biomaterials and condition for measurement of superoxide.

serum did not alter the response of neutrophils to Velcro. The neutrophils (5 X lo5) in a volume of 500 pL KRPG were incubated on the biomaterials for 10,30, or 120 min at 37°C with shaking to allow attachment to the material to occur. After these intervals of association, 200 pM cytochrome c with and without activators (fMLP M and PMA 1 pg/mL) were added. The final volume was 1 mL in quadruplicate samples where the fourth well contained 50 pg/mL SOD. The reaction was allowed to continue for 30 min at 37°C with shaking at 100 oscillations per minute. The plates were placed in melting ice

KAPLAN ET AL.

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to stop the reaction and then were immediately centrifuged at room temperature at 750X g for 10 min. After centrifugation, 0.2 mL of the cell-free supernatants was then transferred to 96-well microtiter dishes for determination of the amount of cytochrome c reduced during the 30 min of incubation just described. The amount of SOD-inhibitable 02-released was calculated as described above. Data are expressed as 02-release per lo6 neutrophils/30 min and the effect of the biomaterials and the times of incubation were compared.

Statistical analysis The effects of interest were differences between biomaterials, effect of time with each stimulus, and interaction between the biomaterials and time. For study 1, a repeated-measures analysis of variance (ANOVA) was used to analyze the data. Following a significant omnibus effect, Scheffe’s posthoc proced ~ r was e ~used ~ to examine pairwise comparisons. All statistical assumptions were met for the univariate model. These were performed for each outcome separately (none, fMLP, and PMA). In addition, for study 2 a multivariate repeated-measures ANOVA was used since the assumptions for the univariate analysis were not met. The biomaterials were compared to each other and the data were analyzed considering the effect of time on the response to each biomaterial and for each stimulus with each biomaterial (none, fMLP, PMA).

RESULTS

Reactivity of neutrophils incubated with and then removed from biomaterials The amount of 02-produced by neutrophils following removal from polystyrene, polyurethane, and woven dacron after periods of contact of 0, 30, and 60 min is shown in Table I. In the absence of an additional stimulus, none of the materials induced a statistically significant alteration of neutrophil activity. The cells that had been on polyurethane and woven dacron, however, showed slightly more activity after a prior 30 or 60 min of association with the materials when compared to the activity of cells removed after 0 min of association. When the neutrophils were stimulated with fMLP, there was significantly less 02-produced by cells that had been on polystyrene and polyurethane for 60 min when compared to the activity of cells that had been in prior contact for 30 min. The effect on 02-release of removal after 30 min of association with the biomaterials was variable. In the seven studies on polystyrene, a twofold rise in response to fMLP was seen in three experiments, no effect was seen in three, and a decrease in response was seen in one. In 10 studies with polyurethane, a 1.6-fold enhancement of reactivity was seen in six experiments while minimal change was seen in the remaining four studies. In contrast, a statistically significant enhancement of response to

6 6 4

30 60

n 0

30

Polyurethane Incubation Time (min) 60 n

0

30

1.4 3 0.4 21.9 2 2.7d,f 52 2 8.6

60

Woven Dacron Incubation Time (min)

0.6 2 0.3’ 0.3 2 0.1 0.5 2 0.1 10 0.2 2 0.05 0.7 2 0.3 0.6 f 0.2 10 0.5 2 0.1 1.3 ? 0.3 13.2 t 3.1 16.9 t 3.1 11.6 2 3.6d 10 15.2 t 2.1 20.3 2 3.0 13.3 t 2.2d 10 15.7 2 3.1 26.0 2 3.6‘ 48 f 5.4 57 ? 7.1 48 t 6.5 6 48 t 4.7 62 2 8.4 37 ? 4 33 2 6.6 10 40f9

0

a n m 02-/10hneutrophils +- SEM. bNumber of studies. ‘Ten-minute generation of 02-. d p < 0,0001 comparing 30- and 60-min time periods. e p < 0,0001 comparing 0- and 30-min time periods. p < 0.0001 comparing 0- and 60-min time periods. gFive-minute generation of 02-.

None‘ fMLP‘ PMAg

Cell Stimulant nb

Polystyrene Incubation Time (min)

TABLE I Superoxide Release” by Human Neutrophils Exposed to Several Biomaterials for Various Time Intervals Prior to Evaluation

KAPLAN ET AL.

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fMLP was observed in the case of exposure to woven dacron for 30 min when compared to the response after exposure for 0 min. Here, 10 of 10 studies provided similar results. While a longer exposure to woven dacron resulted in a drop in reactivity to fMLP, the response was nevertheless higher than that of cells removed after 0 min. In contrast to the enhanced stimulated response to fMLP, the response to PMA was not affected by a prior exposure to biomaterials.

Reactivity of neutrophils while in contact with the biomaterials (in situ) The results of this group of experiments are shown in Tables 11-V. In the absence of an additional stimulus, we observed that polyurethane, Velcro, and woven dacron activated neutrophils and that the amount of activation was dependent upon the material: woven dacron > polyurethane > Velcro > TABLE I1 Superoxide Release”by Human Neutrophils in Contact with Biomaterials without Additional Stimulation

Time on Biomaterials (min) Material

nh

Polystyrene Polyethylene Velcro Polyurethane Woven dacron

13 2 11 5 6

10 1.9 4 9.8 31 48

0.4

-C

120

30

C 2.0’ t 11‘ t- 11‘

1.4 t1.5 6.3 t14 C 44 C

0.3 1.2d

5.0d 9.6

1.6 ? 0.7 1.1 1.6 t 0.5’ 2.3 t 1.0’ 10 ? 2.9’

a n m 02-/106 neutrophils t SEM. bNumber of studies. ‘ p < 0.0001 compared with polystyrene. d p < 0,0001 compared with 10 min. ‘ p < 0,0001 compared with 30 min. TABLE 111 Superoxide Release”by Human Neutrophils in Contact with Biomaterials and Stimulated with FMLP

Time on Biomaterials (min) Material

nb

Polystyrene Polyethylene Velcro Polyurethane Woven dacron

13 2 11 5 6

10 13 2 16 15 5 38 t62 5

nm 02-/106 neutrophils ? SEM. Number of studies. ‘ p < 0.0001 compared with 10 min. d p < 0.0001 compared with 30 min. e p < 0.0001 compared to woven dacron. f p < 0.0001 compared with polystyrene.

a

1.5 2.3’ llf

13‘

30 9.2 C 1.2’ 5.7‘ 8.6 t 1.8’ 20 -c 3.6‘ 61 C 13

120 6.1 t5.7 3.4 t5.1 t22 -t

1.8d 1.0d 0.6d 4.84

BIOMATERIAL-INDUCED NEUTROPHIL ACTIVATION

1047

TABLE IV Ratio of Superoxide Released-Stimu1ated:Unstimulated"

Time on Biomaterials (min) 10

30

120

Material

nb

fMLP

PMA

fMLP

PMA

fMLP

PMA

Polystyrene Polyethylene Velcro Polyurethane Woven dacron

13 2 11 5 6

6.8 4.0 1.5 1.2 1.3

63 17 5.1 2.5 3.1

6.1 3.8 1.4 1.4 1.4

78 38 5.2 5.4 3.3

5.5 5.2 2.1 2.2 2.2

33 43 8.1 21 11

a Calculated as follows: 02-nm/106 neutrophils of fMLP- or PMA-stimulated cells divided by 02-released by unstimulated cells. Number of studies.

TABLE V Superoxide Releasea by Human Neutrophils in Contact with Biomaterials and Stimulated with PMA

Time on Biomaterials (min) Material

nb

Polystyrene Polyethylene Velcro Polyurethane Woven dacron

13 2 8 5 6

10 120 2 67 50 ? 78 2 147 ?

30 10

7.F' 4.3'*' 14'

109 ? 11 57' 33 ? 7.0' 75 2 5.3 144 t 13'

120 52 ? 47d 13 ? 48 ? 105 ?

13cfd 4.1d 6.Fd llC,d

a n m 02-/106 neutrophils 5 SEM. bNumber of studies. ' p < 0.0001 compared with 10 min. d p < 0.0001 compared with 30 min. ' p < 0.0001 compared with polystyrene. * p < 0.0001 compared to woven dacron.

polystyrene/polyethylene controls (see Table 11). The degree of activation also was dependent upon the duration of contact with the biomaterials-10 min > 30 min > 120 min with polyurethane and Velcro -while woven dacron had a more persistent activating influence: 10 min = 30 rnin > 120 min. We note, however, that even after 120 min of biomaterial contact the 02-release was not less than the release of cells on nonactivating polystyrene and polyethylene. Serum-coated polystyrene did not activate the neutrophils regardless of the length of contact time, and 02-production was less than 1.9 nmol per lo6 cells. Additional stimulation of biomaterial-associated neutrophils with fMLP resulted in a greater amount of 02-release than was induced by the biomaterials alone. These data are shown in Table 111. The greatest amounts of 02release occurred from neutrophils on woven dacron and polyurethane (woven dacron > polyurethane). Incubation on Velcro was not associated with an enhanced response to fMLP when compared to the nonactivating controls, coated polystyrene and polyethylene. A time-dependent lessening of response to fMLP occurred on all the materials, following the pattern:

KAPLAN ET AL.

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10 min > 30 min > 120 min on polystyrene, Velcro, and polyurethane; 10 min = 30 min > 120 min on woven dacron; and 10 min > 30 min = 120 min on polyethylene. Incubation on polystyrene, polyethylene, and velcro for 10 min followed by fMLP stimulation was associated with similar although the ratio of activity of stimulated to unstimuabsolute levels of 02-, lated cells was less with Velcro because the unstimulated activity was higher (Table IV). Although PMNs on polyurethane and woven dacron displayed the greatest 02-release, the ratio of fMLP-stimulated 02-to unstimulated 02-was also quite low. Interestingly, while the absolute level of response decreased over time, the ratios of stimulated to unstimulated activity changed relatively little with all materials. The effect of stimulating neutrophils on biomaterials with PMA is shown in Table V. The cells on woven dacron were more responsive than those on any of the other materials, followed by the cells on polyurethane and Velcro. Neutrophils on Velcro and polyurethane, however, were less responsive than those on the coated polystyrene control. Decreasing responsiveness with longer incubation was observed but the usual response pattern with PMA stimulation was 10 min = 30 min > 120 min. The exceptions were Velcro and the control polyethylene, where the response pattern was 10 min > 30 min > 120 min.

DISCUSSION

These studies have shown that when neutrophils remain in contact with a variety of biomaterials they readily become activated and release 02-.This occurs when cells were evaluated in the absence of an additional activator, after stimulation with the receptor linked activator fMLP, or after the direct stimulation of protein kinase C with PMA. The degree of activation was related to the duration of contact time with the materials, where shorter periods of contact were associated with more 02-release than occurred with longer periods of contact. This was especially evident with unstimulated cells and after fMLP stimulation. The responsiveness to PMA was more persistent but also diminished with time. While decreasing responsiveness with increasing in situ exposure time could simply be due to age-related loss of reactivity or viability, we doubt this is the case since neutrophils held in test tubes retain undiminished reactivity and viability for many hours (data not shown). Neutrophils, however, are end-stage cells with limited capacity for renewal. It is more likely, therefore, that biomaterial-induced activation results in progressive depletion of proteins involved in the oxidative burst, including granule constituents that may be released with the biomaterialinduced activation. While this might be expected to seriously impair host defense to microbial invaders, we observed that even the cells with the smallest amount of reactivity were at least as reactive as the cells on the control materials. These data, together with maintenance of reactivity to PMA, suggest that the neutrophils’ capacity for host defense may be diminished but not entirely lost.

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Consistent with the idea that the neutrophil has limited resources was the observation that the cells exhibiting the greatest production of free radicals in the absence of any additional stimulus also had the least additional response to fMLP. However, the cells remained more responsive to PMA, a nonphysiologic chemical probe that induces activity beyond the level of regulation at the cell surface. Thus, the neutrophils' capacity for free radical production appears less affected when this probe is used. Alternatively, there may be other families of receptors that remain responsive via PKC activation. Since it is likely that it is surface contact that induces activation, it would be expected that woven dacron, a fabric with a large amount of surface area, would be more activating than polyurethane, which is fairly smooth. This cannot be the entire explanation for the high level of activity associated with woven dacron, however, because Velcro also has a large surface area and it did not appear to be as stimulatory as polyurethane. To exclude that Velcro may interfere with cytochrome c measurement of 02-formation, we evaluated the effect of Velcro on xanthine/xanthine oxidase-induced 02-formation and found no diminution of 02-(data not shown). It should be considered, however, that with prolonged association of neutrophils with some biomaterials much cell activation could be occurring into a tightly closed compartment as had been observed by Heiple et al. with macrophages in close apposition to IgG-coated surfaces.28 The studies in which neutrophils were exposed to biomaterials and then removed demonstrated neither increased unstimulated activity nor altered response to PMA. With fMLP, however, cells that had been on woven dacron for 30 or 60 min exhibited an increased response. In contrast, the cells removed from polystyrene and polyurethane displayed lessened responsiveness to fMLP after 60 min compared to 30 min of exposure. These neutrophils, however, most likely had a briefer encounter with the biomaterials compared to the studies performed in situ since these were the cells that could be easily removed from the materials. Thus, the degree of biomaterial influence was likely to be quite variable within and between studies. The data does suggest, however, that a neutrophil that encounters a biomaterial but does not attach to it may not be appreciably altered. Thus, for the short time periods considered in this study functional lessening, but not obliteration of response, occurred and the early infectivity around biomaterials may require an alternative explanation. All our periods of observation are short, however, when placed in the perspective of long-term implantation in vivo. These observations are consistent with the reported findings of Zimmerli et a1.I' using an implanted Teflon cage in an animal model and aspirating cells and fluid 14 days later. These cells exhibited a profound functional deficit and animals were easily infected unless fresh neutrophils were introduced near the time of infection, and then again later. Thus, neutrophils that were less able to respond to mediators of inflammation resulted in impaired host resistance to local infection. A study of longterm biomaterial implantation is necessary to determine whether the observed effects of biomaterials on neutrophils persist or whether the early interaction of materials and cells provides for a less interactive surface when later cells arrive. It must also be considered, however, that an early infection

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KAPLAN ET AL.

that was not adequately addressed provides for an intracellular focus of persistent (though possibly inactive) infection that may become active when conditions in favor of the parasite become predominant. This work was supported by NIH Grant R01-GM41734-01. The authors acknowledge the excellent technical assistance given by Myung Hee Jeong and the typing performed by Lorraine Herr and Jasmine Parisi. Dr. Janine Janosky performed the statistical analysis.

References 1. J. Charnley, Postoperative infection after total hip replacement with special reference to air contamination in the operating room. Cliiz. Orthop. Relat. Res., 87, 167-187 (1972). 2. M. Levine, S. J. Rehm, and A. H . Wilde, Infection with Candida albicans of a total knee arthroplasty. Clin. Orthop. Relat. Res., 226, 235-239 3. 4.

5. 6.

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(1987). T. Parthasarathy and S. Rajeswari, Failures in orthopaedic implants. lnd. I. Med. Res., 84, 440-443 (1986). B. Almgren and I. Eriksson, Local treatement of infected arterial grafts. Acta Chir. Scand. Suppl., 529, 91-94 (1985). J. E. Rosenman, W. H. Pearce, and R. F. Kempczinski, Bacterial adherence to vascular grafts after in ziitro bacteremia. 1. Suug. Res., 38, 648655 (1985). T.M. Bergamini, D.F. Bandyk, D. Govostis, H.W. Karbnick, and J.B. Towne, Infection of vascular prostheses caused by bacterial biofilms. 1. Vasc. Surg., 7, 21-30 (1988). D. E. Szilagyi, R. F. Smith, J. P. Elliot, and M. P. Vrandecic, Infection in arterial reconstruction with synthetic grafts. Ann. Smug., 176, 321-333 (1972). V.M. ’Bernhard, Management of infected vascular prostheses. Surg. Clin. N. Am., 55, 1412-1417 (1975). A.G. Gristina, J. J. Dobbins, B. Geammara, J.C. Lewis, and W.C. DeVries, Biomaterial-centered sepsis and the total artificial heart-microbial adhesion versus tissue integration. JAMA, 259, 870-874 (1988). W. Zimmerli, P. D. Lew, and F. A. Waldvogel, Pathogenesis of foreign body infection. Evidence for a local granulocyte defect. 1. Clin. Invest., 73, 1191-1200 (1984). A.G. Gristina, Biomaterial centered infection: Microbial adhesion versus tissue integration. Science, 237, 1588-1595 (1987). S.H. Dougherty and R . L . Simmons, Infections in bionic man: The pathobiology of infections in prosthetic devices. Curr. Prob. Surg., l’art 1, 19, 221-264 (1982). B. Sugarman and E. J. Young, Infections associated with prosthetic devices: Magnitude of the problem. Inf. Dis. Clin. N. Am., 3, 187-198 (1989). E. J. Young and B. Sugarman, Infections in prosthetic devices. Surg. Clin. N.Am., 68, 167 (1988). R. D. Zipser and W. L. Henrich, Implications of nonsteroidal antiinflammatory drug therapy. A m . J. Med. Suppl., l A , 78 (1986). W. Zimmerli, F. A. Waldvogel, P. Vandaux, and V. E. Nydegger, Pathogenesis of foreign body infection: Description and characteristics of an animal model. J Infect. Dis., 146, 487 (1982). B. H. Wade and G. L. Mandell, Polymorphonuclear leukocytes: Dedicated professional phagocytes. Am. J. Med., 74, 686-693 (1983).

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Received December 24,1990 Accepted December 23, 1991