Technical note

Platelet consumption by NHL BI reference materials and Silastic W. F. Ip and M.V. Seftod Department of Chemical Engineering and Applied Chemistry and Centre for Biomaterials, University of Toronto, Toronto, Ontario, Canada M5SlA4

IN T RODUC T ION

Assessing the platelet reactivity of a biomaterial is part of determining its thrombo- genicity. While platelet adhesion measurement or SEM analysis of adherent platelet morphology addresses this issue in part, it is difficult to extrapolate from these mea- sures to the effect on platelets that may make only transient contact with the surface, or which may be removed from the surface as emboli. This has been particularly impor- tant for at least certain hydro gel^,'-^ where platelet adhesion has been low but platelet consumption has been high. High platelet consumption in the absence of significant platelet/thrombus deposition may not be confined to just hydrogels. Hanson et al. reported high consumption rates for Biomer and other polytetramethyleneglycol- polyurethanes with a high surface ether content! Examination of the residue on the surface (adherent platelets or thrombotic deposits) is not sufficient to conclude that a material has a low thrombogenicity. An assessment of consumption or embolization is required as well.

Here we report on platelet consumption by the NHLBI reference materials and Silas- tic, in the dog, using a chronic shunt. Platelet consumption was assessed indirectly by measuring platelet regeneration using the malondialdehyde (MDA) a ~ s a y . ~ , ~

MATERIALS A N D METHODS

NHLBI reference materials were obtained from Thoratec (silica free polydimethyl siloxane, PDMS, Berkeley, CA) and Abiomed (polyethylene, Danvers, MA) as 4-mm-I.D. tubes. Silastic tubes (3.18 mm I.D., Dow-Corning, Midland, MI) were also tested. Tubes were inserted between arterial and venous cannula portions of a chronic arteriovenous previously established in a dog.* For the 4-mm-I.D. tubes (reference materials), adapters were fabricated from Silastic tubing to enable them to be connected to the 3-mm-I.D. cannulae of the shunt. The day prior to tube insertion the animal was given aspirin to inhibit irreversibly platelet cyclooxygenase. As new platelets were produced, the cy-

*To whom correspondence should be addressed.

Journal of Biomedical Materials Research, Vol. 25, 1321-1324 (1991) 0 1991 John Wiley & Sons, Inc. CCC 0021-9304/91/101321-04$4.00

1322 IP AND SEFTON

clooxygenase activity returned to normal baseline levels, such activity being measured by the in vifro production of malondialdehyde by a sample of platelets taken each day (for 4 days) from the animal. The decrease in platelet count and increase in MDA pro- duction were combined to calculate the initial fractional rate of platelet destruction.’ The assumptions in this method are explained e l~ewhere .~

RESULTS AND DISCUSSION

The platelet counts are shown in Figure 1. There was little difference between the reference materials and the corresponding commercial material, although the consump- tion rate (Table I) for the reference polyethylene was greater, albeit not significantly, than for Intramedic polyethylene (reported before in Ref. 3). The thrombocytopenia for the two silicones was intermediate between that for polyvinyl alcohol (PVA) and the polyethylenes. The reference materials and polyurethane lead to a degree of con- sumption intermediate between that for Intramedic polyethylene and PVA.

Hanson and Harker’ found Silastic to be one of the least reactive of the materials they studied, but still more reactive than Intramedic polyethylene, consistent with our results. However the reactivity of the silicone test sections is not consistent with the low consumption of the Silastic shunt without a test section (i.e., the blank). The con- sumption rate was not measured in the absence of the shunt because of experimental difficulties. However, the -1 m of Silastic tubing used for the AV shunt must not be

I 0 PE-reference PDMSreference 0.2 1

0 0 - 1 ----d___i__

A Sltastlc -.-. PVA ------.polyethylene (Intramedic)

I ] -2-

Time (days)

0

Figure 1. Relative platelet count during test section exposure to canine blood in AV shunt. Test section inserted on day 1. D, polyethylene reference; ., PDMS reference; A, Silastic. Mean 2 SD from three individual experi- ments in three separate animals. ---, Intramedic polyethylene; PVA (both from Ref. 3); there was no platelet count decrease with a shunt only blank.

PLAT EL ET C ONSUMP TION 1323

TABLE I Initial Platelet Consumption Rates

Material %/Day a p < 0.05b

Polyethylene-ref erence PDMS-reference Silastic Blank (no test section)' Intramedic polyethylene' PVA'

27.1 ? 0.4 (3) 28.7 ? 1.7 (3) 26.0 rf: 4.8 (3) 18.0 ? 3.0 (15) 20.5 ? 5.0 (12) 34.0 k 4.5 (10)

Yes Yes Yes No"

Yes -

aBased on mean of initial slopes of individual consumption curves measured in at least 3 animals. -+ standard deviation among individual values (n given in brackets).

bSignificance relative to Intramedic polyethylene at 0.5 level (ANOVA, standard de- viation = 4.34 based on 46 degrees of freedom within groups including data in Ref. 3).

'Reported previously (Ref. 3). " p < 0.1, relative to Intramedic polyethylene.

very reactive since the -17%/day consumption rate (blank) is consistent with the nor- mal 5.4-6.9-day canine platelet lifespan and since it is possible to distinguish the effect of an additional 20 cm of test tubing. The difference between the silicone shunt and test section may reflect a flow effect associated with the change in tube curvature when the test section is in place or a difference between fresh Silastic (test section) and that which has been exposed to blood for several weeks or months (shunt).

The results presented here indicate that the NHLBI reference materials were more reactive toward platelets than the Intramedic polyethylene control. These observations are not inconsistent with those of McCoy et aL5 on canine platelet deposition in a simi- lar AV shunt. With an acute shunt, platelet deposition reached a maximum of -150- 200 platelets/1000 pm2 near 30 min on both reference materials with a similar pattern on Intramedic polyethylene; a smaller maximum was seen on Silastic. Similar patterns were seen at a lower shear rate (670-800 s-', instead of 1350 s-') in a chronic shunt adapted from the one used here. However, deposition values were somewhat higher (-500-800 platelets/1000 pm') and Silastic was comparable to the silicone reference material and the reference polyethylene ranked slightly better than the Intramedic polyethylene.

Pelzer and Heimburger6 measured in vifro (Chandler loop) human platelet factor 4 release (by immunoassay) and ex vivo human fibrinogen and platelet deposition using rabbit anti-human antisera with the reference materials and found parallel behavior in both systems. The reference polyethylene induced high levels of PF4 release and took up higher amounts of the antibodies, suggesting a more platelet activating surface than the reference silicone.

The authors acknowledge the financial support of NIH (HL24020) and Dr. Walter Zingg and his staff.

References 1. S. R. Hanson, L. A. Harker, B.D. Ratner, and A.S. Hoffman, "In vivo

evaluation of artificial surfaces with a nonhuman primate model of ar- terial thrombosis," J. Lab. Clin. Med., 95, 289-304 (1980). C. H. Cholakis, W. Zingg, and M.V. Sefton, "Effect of heparin-PVA hy- drogel on platelets in a chronic canine AV shunt," 1. Biomed. Muter. Res.

2.

23, 417-441 (1989).

1324 IP AND SEFTON

3. W.F. Ip and M.V. Sefton, "Platelet consumption by polyvinyl alcohol coated tubing in canines," J. Biomed. Mater. Res., in press.

4. W. Zingg, W. F. Ip, M.V. Sefton, and K. Mancer, 'A chronic arteriovenous shunt for the testing of biomaterials and devices in dogs," Life Szcpp. Syst., 4, 221-229 (1986). T. J. McCoy, T.G. Grasel, A. Z . Okkema, and S. L. Cooper, "Acute and chronic canine ex vivo blood interactions with NHLBI-DTB primary reference materials," Biomatcriak, 10, 243-250 (1989). H. Pelzer and N. Heimburger, "Evaluation of the in vitro and ex vivo blood compatibility of Primary Reference Materials," J. Biomed. Muter.

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6.

RE., 20, 1401-1409 (1986).

Received March 27, 1993 Accepted April 17, 1991