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THE ROLE OF SURFACE ENERGY IN THROMBOGENESIS*


ROBERT E. BAIER, Ph.D.
Principal Physicist Cornell Aeronautical Laboratory of Cornell University Buffalo, N.Y.

T HE present situation with respect to the evaluation of biomedical materials might be likened to that of a large number of individuals all busily exploring mechanisms for exit from their own independent circular mazes. After final breakthrough of the walls of any independent circular maze we discover only that we have all along been within a much larger, more intricate maze-the whole complex maze of biomedical problems-and that there are many other investigators still within isolated circular loops like the one from which we have recently emerged. Figure I illustrates an overview of three such confining rings, one labeled surface texture; one, surface charge; and the other, surface chemistry. These rings represent three of the primary surface characteristics that are now being carefully examined in the hope of discovering "magic numbers" which might apply in determining the ultimate thrombogenicity or thromboresistance of a candidate biomaterial. The figure takes the optimistic option of showing regions where these three circles may overlap. Stated in the simplest possible terms, the general goal of many of the investigators contributing to this program is to find that specific area where their own special interest, their own "magic number" if you will, overlaps any or all of the others. Our common goal is eventually to provide the biomedical engineer and the general medical community with a specific set of parameters that can guide them in the formulation of new and better blood-contacting materials. Still, it is recognizable that the surface qualities of the materials are not the only factors to be considered. They can be contrasted with all of those
*Presented as part of a Symposium on Problems in Evaluating the Blood Compatibility of Biomaterials held by the Section on Basic Medical Sciences and the Section on Biomedical Engineering of the New York Academy of Medicine February 18 and 19, 1971. This study was based upon research internally supported by Cornell Aeronautical Laboratory, Inc.

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properties which are intrinsic to the blood volume itself, as indicated at the right side of Figure i. The surface characterization approach is often at odds with the "volume" approach where considerations such as flow, blood coagulation factors, and yet unknown factors are given primary consideration. Details of the probable role of some of these "volume" effects will be given in other presentations at this conference. Before limiting our attention to the role of surface energy in the determination or evaluation of potential thromboresistance of materials let us consider the alternative mechanisms that have been proposed for obtaining thromboresistance. Figure 2 lists eight different concepts presently receiving consideration. On the left and right of this figure, in the darkly outlined boxes, are listed the various approaches that different groups are using. For instance, very smooth surfaces, negatively charged surfaces, low critical surface tension materials, and materials with hydrated layers have all been proposed for controlling thrombus formation. The vertical trunk running down the center of Figure 2, with the solid large arrows pointing to boxes on the left, indicate that at least in some quarters claims have been made that the living blood vessel intima has these indicated properties. Under each of
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these boxes are concepts or materials that are presently being looked at for mimicking each of the qualities. For example, for very smooth surfaces we are considering polished metals or glasses. Active exploration of negatively charged surfaces involves investigations with metals holding various rankings in the electrochemical series and with solidified polyelectrolytes. ' 2 For low critical surface tensions, some groups have looked at waxy layers and fluorocarbons. For hydrated layers, many laboratories are now looking at the hydrophilic polymers and even at components in which hygroscopic salts are present that can accrete moisture and therefore bind and organize a watery interfacial layer. On the right hand side of Figure 2 are some additional approaches which have not been given sufficiently serious attention as potentially characterizing living blood vessel intima. Since they may eventually receive such attention, dotted arrows connect the vertical trunk in the center of Figure 2 to these surface characteristics. Among ablative surfaces, it is believed, for example, that heparinized materials, albumin-coated materials, and those polymers which are doped with surfactants may exhibit thromboresistance because of simple erosion of their surface layers and concomitant anticoagulation of the boundary layer of blood.' The "natural materials" now being considered include heterografts of bovine origin;4' 5there are also new and exciting prospects of culturing living
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endothelial cells on textured materials of such nature that flowing blood will "see" only the compatible endothelial cell surfaces it has been used to "seeing" in the natural environment. Other concepts include the introduction of slippery layers at biomaterials interfaces; one might inject a drag-reducing polymer into the flowing blood, or create implant materials whose surfaces are slowly solubilized. Yet another approach, in extreme contrast to the low critical surface tension approach indicated at the left of Figure 2, is that of selecting an extremely highsurface-energy material. Long-term patency has been noted with materials such as glow-discharge-treated metals and glasses and with intrinsically high-surface-energy pyrolytic carbons available from the Gulf General Atomic Corporation.3' Now let us consider more carefully the single parameter of a biomedical material surface which we believe holds the greatest promise for becoming a generally-specified characteristic: the critical surface tension. The table shows a simple ranging of the critical surface tensions, Ye, of common plastics. The experimental methods by which these values are obtained have been presented elsewhere.7 8 One can see quickly that each common plastic, indeed each common solid surface, has associated with it a "magic number" called critical surface tension. This parameter is expressed in dynes/cm. or ergs/cm.2, an energy term ranging from about i 8 dynes/cm. for Teflon through the mid-30 dynes/cm. range for polyethylene-based materials all the way up to 46 dynes/cm. for common nylon. Thus we have available a parameter which has proved to be characteristic of only the solid surface and not of any techniques used to obtain it.7 8 The critical surface-tension parameter varies over a very broad range, can be easily assessed, and may be correlated with biomedical interactions. Why is the critical surface-tension parameter, related to surfacefree energy, worthy of attention as a surface characteristic which might control the intrinsic thromboresistance or thrombogenicity of a biomedical material? An early answer, provided in what might be considered ancient history in the field of biomedical material evaluation, appeared in a 1958 study of then-available materials implanted as vascular grafts.9 After different times of implantation, generally 30 to So days, they were excised and the graft surfaces were inspected. When nylon grafts were opened, there was the general finding of a very thick fibrin lining on the inner surface and of a large superimposed thrombus
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which completely occluded the lumen of this material. When a series of implanted graft materials were examined after excision and transection to reveal their cross section, it was found that after 3o days in vivo nylon grafts acquired a very thick luminal lining, Mylar a slightly thinner luminal lining, but Teflon had only a thin inner and outer capsule. If the critical surface tensions for these materials had been examined at that same time, one would have immediately noted the apparent correlation between the critical surface tensions of the materials and their coating with biological products from the bloodstream. Nylon has the highest critical surface tension, and Mylar the next highest, the lowest in this particular series being Teflon. To cite another example: consider recent biomedical research on artificial skin.'0 In Figure 3 is a ranking of the relative tissue adhesion noted for the materials rayon, nylon, Dacron, polypropylene, and Teflon. Similarly ranked in this figure are the relative critical surface tensions for these same materials as determined from contact-angle studies of W. A. Zisman and his associates over the past three decades.7 Here, again, we see an apparent direct correlation with the relative surface energy of the material. In those cases where relatively strong tissue adhesion was observed for nylon and Dacron, we were also confronted with a high critical surface tension. For situations where the lowest relative tissue adhesion was observed, with polypropylene and Teflon, we also noted a lower critical surface tennsion. So here, in a totally different biomedical specialty, is an example of how this apparent "magic parameter," the critical surface tension, has correlated with biological adhesion. In a study of very direct relevance to the present subject, Lyman, Brash, and their colleagues showed that platelet adhesion to various surfaces could also be correlated with the critical surface tensions of those surfaces." It was found that, for very short contact times, there was an apparent direct relation between the critical surface tension of the material and the average number of adherent platelets. We have since learned from studies by Dutton and his co-workers, 12 by Friedman and his co-workers'3, and by Petschek and his co-workers14 that the differences in this initial platelet adhesion tend to be masked with increasing blood contact time: every surface eventually accumulates the same apparent saturated density of platelets regardless of its original critical surface tension, and yet these materials have different ultimate fates during long-term implantations. It is interVol. 48, No. 2, February 1972

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esting that Lyman and his group could also demonstrate an inverse correlation between the critical surface tension of a material and its blood clotting time.'5 When the critical surface tension of the material was high the blood clotting time was very short; when the critical surface tension of a polymer was very low, then the clotting time, in a simplified test, was elongated.15 So here we have another situation where the severity of the interaction with the blood seems to have been dictated by the critical surface tension of a candidate biomedical material. We can move directly now to an example of pertinence to the most common of biomedical implants, substitute heart valves. In many of the heart valves that have been implanted, the basic construction of the firm structural components: that is, of the ball-valve cages, the struts, and valve seats, has used the metal alloy Stellite 2i. There was no special reason why Stellite, a simple alloy of cobalt and chrominum mainly, should have shown this apparent thromboresistance. However, when contact angle techniques were used to assess the critical surface tensions of these materials, we were able to demonstrate that those surfaces which were thromboresistant were surfaces that had accumulated, during the final stages of manufacturing, a coating of hydrophobic contaminant.'6 An adventitious manufacturing contaminant generally derived from tallow had been lapped into the surface of the Stellite and therefore the blood stream was confronted not with the metallic surface at all but with an organic surface whose critical surface tension was in the range of about 25 dynes/cm. We were able to show, further, that as one stripped this adventitious layer, one obtained higher critical surface tensions, relatively stronger interactions at the solid/liquid boundary, and also increasing thrombogenicity.'6 From a variety of ancillary studies we were able to isolate a critical surface tension range of some 20 to 3o dynes/cm. as probably more biocompatible than other ranges. The utility of this hypothesis is nicely confirmed by a completely independent study of the adhesion of single cells, in tissue culture, to various substrates.'7 If one plots the critical surface-tension data for the substrates to which cell attachment occurred one finds that there was a zone of minimal interaction, that is, of minimal cell attachment and cell spreading, in the region between 20 and 3o dynes/cm.; it actually centered again on some 25 dynes/cm.'8 We can then label this zone of minimal cell spreading, or zone of minimal cell interaction, as a hypothetical zone of biological compatibility.'8 Having correlated in
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the past the critical surface tensions of materials with the actual outermost atomic constitution of the surfaces, we are able to say that this general critical surface-tension range represents surfaces almost totally dominated by closely packed methyl groups.8 18 Figure 4 represents our tentative correlation of the relative surface energies of solids with their biological interactions and represents a general summary of a large amount of information, a great deal of which has not been given here, that relates interactions of various types of implants in various locations of the body. We have labeled at the dip of this curve a hypothetical zone of biocompatibility for the region 20 to 30 dynes/cm. Above 4o dynes/cm. we have labeled a zone of good bioadhesion. Those biomedical specialists interested in orthopedic implants, and therefore interested in obtaining good biological fixation of an implant, would be advised to choose a material with a relatively high surface energy. High critical surface tensions should promote very strong interactions with the biological phase, including promotion of good cell spreading and attachment. For instance, in the use of a porous ceramic or a porous metallic sponge material, good cell infiltration and perhaps recalcification should be observed. One will note from this diagram, as well, that there is a very narrow zone which we have labeled as including most common polymers. With the exception of fluorocarbon type materials, the majority of commercially available plastics fall into this zone, this nebulous zone of critical surface tensions between 3o and 40 dynes/cm. Thus readily available plastics are neither good as biocompatible materials nor as substrates for biological fixation. Our dilemma today is that most of these presently available polymeric materials for biomedical engineering have neither valuable property when assessed by the surface-energy approach. At the very high energy extreme of Figure 4 there is a hypothetical falling off of the plot to show that there is a potential zone of very good biological compatibility for extremely high energy surfaces. This was alluded to earlier, and is labeled in Figure 2 as one of the approaches being considered for imparting thromboresistance. For example, glowdischarge-treated, extremely high surface energy, common commercial Pyrex glass implanted in the inferior vena cava has been staving essentially thromboresistant for long periods of time.'9 The low critical surface-tension approach, although definitely quite useful and interesting, certainly is not the only possible approach and not the only way to
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Fig. 5. Reproduced by permission from: Baier, R. E. and DePalma, V. A.: Management of Occlusive Arterial Disease, Dale, W. A., editor. Chicago, Year Book Med. Publ., 1971.

design new biomaterials from a surface-energy viewpoint. An important complication that must be considered when one is attempting to define biomedical interactions or relative thrombogenicity in terms of surface energy is the influence on the interactions with the biomedical phase of simple surface toughness: that is, of differences in surface texture. For example, we have known for a long time in our studies of the alloy Stellite 2 I, that if one sufficiently roughens the surface, eliminating the adventitious waxy contaminant that has been responsible for the good surface chemistry or even without changing the surface chemistry, the induction of roughness in the surface over which blood must flow can lead to a complete lumen-blocking thrombus. One is very much interested in determining what kind of parameters can be assessed in determining the roughness on both a microscopic and a macroscopic scale and how these surface texture parameters might overlap with the surface-energy parameter. Figure 5 is an electron micrograph of a replica of the internal surface of one of the newer heterografts of bovine origin that have been having extremely good success during human implantation.20 One can see from this figure that the surface texture is remarkably rough, the whole smooth internal lamina having been digested away so that only the collagenous matrix is left. On a microscopic scale, this is an extraordinarily rough surface, and one might predict an unhappy course of events for this material when implanted in the bloodstream, as a femoralpopliteal bypass graft, for instance.
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Fig. 6. Reproduced by permission from: Baier, R. E. and DePalma, V. A.: Management of Occltsive Arterial Disease, Dale, W. A., editor. Chicago, Year Book Med. Publ., 1971.

Figure 6 shows an identical graft after only 20 minutes of implantation. It shows that the surface has become, comparatively, extraordinarily smooth. We have shown that this occurs by deposition from the blood of a proteinaceous mat probably dominated by fibrin.5 This deposit so smooths over the interior surface of this graft material that, at least after a few minutes of implantation, the surface chemical factors must be controlling and the surface roughness that was originally noted has been disguised completely. Carrying this to a more gross level, we have had the opportunity recently to consider all of the available fabric grafts, as commercially woven and knitted from Dacron and Teflon fibers. These represent the large majority of grafts that are being used in vascular replacement operations today. It has been discovered that in these cases, as well, a covering-over of the intrinsic surface roughness by a layer of proteinaceous material occurs. In particular, the manufacturer's instructions in each case recommend a preclotting routine. That is, the patient's blood is generally taken in a syringe and, prior to implantation of the prosthesis, the blood is injected into the prosthesis and allowed to preclot for a given period of time. Therefore the prosthetic surface that the fresh flowing blood is actually exposed to is neither the Teflon nor the Dacron but really a layer, perhaps a biocompatible layer, that has formed on this material during the preclotting regimen. The difficulty is that this preclotting regimen seldom seems to give complete covering, so that the adverse very low critical surface tension of Teflon or the adverse very high critical surface tension of Dacron shows
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through. One can then have potentially adverse interactions of these substrates with the blood despite the preclotting regimen. Having introduced the possibility that proteins and protein layers deposited in prostheses are perhaps controlling factors in thromboresistance we can consider the generalization that adsorbed proteins are probably the controlling surface species in almost every biological circumstance and ask a very crucial question: How is the influence of the critical surface tension of a polymer propagated through a proteinaceous layer into the volume of the blood where its continuous state of fluidity or its solidification into a thrombotic mass is controlled? We have shown in earlier work that exposure of an organic-free surface to fresh flowing blood for as little as five seconds, which was as rapidly as we could do the manipulations, leads to its complete coating by a very uniform, tenaciously-adherent proteinaceous film and that this protein is very likely to be fibrinogen.2" On a surface where the critical surface tension is high to begin with, the critical surface tension of the film which accumulates also remains reasonably high. In the case of the five-second adsorbed film we discovered a critical surface tension in the range of between 35 and 40 dynes/cm., and we further discovered upon following the course of events at that surface and at similar surfaces by electron microscopic cross sections, that a typical thrombus mass does indeed grow on such surfaces, separated from the original substrates by the very thin, densely staining, proteinaceous layer.22 The focus of attention thus has shifted from the substrates themselves as inducers of thrombogenicity to the substrates as dictators of a special configuration of adsorbed protein molecules that will favor or inhibit the subsequent events, including activation of the clotting mechanism and adhesion of platelets.,' In work that has been described in greater detail elsewhere3 the prospect that protein configuration changes occur when films are adsorbed at interfaces is given primary attention. The most exciting recent finding has been that a globular protein, in solution at physiologic. ionic strength, can adsorb with varying degrees of denaturation of the native protein molecules on surfaces of high and low surface energy.23 For instance, the ratio of native to denatured protein adsorbed onto a high energy surface will be no more than 4/5, while on a substrate with intrinsically low surface energy it can be up to one. The tentative conclusion can be drawn that the mechanism by which a low critical surface-tension material assists in developing a passive intimal layer on an
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implant is that it allows adsorption of proteins in less configurationallyaltered states than would otherwise occur. In summary: we believe that the surface attraction by the solid influences first and foremost the adsorption of proteins, and especially the configuration of those protein molecules that are adsorbed. Secondarily, this influence is transmitted to the initial adhesion of platelets and perhaps controls the adhesional lifetime of the platelet aggregates which may inevitably form. One proposed mechanism for the surface of an implant remaining clear of thrombotic material is that the lower critical surface tension of the substrate favors a poor general adhesion of the adsorbed protein film, because these adsorbed molecules are more native and more likely to be in pseudoequilibrium with materials in the flowing blood. This leads to a shorter adhesional lifetime of the adsorbed film and of any platelets that it has accumulated, so that the normal shear rates that are encountered at an implant surface will lead to a ready detachment of the thrombus or the thrombus precursors. If this is the case the aggregates that are detached will be so small that they can be handled by normal enzymatic and digestive processes in the flowing stream, and one has a decreased likelihood of an adverse thromboembolic event some place downstream. Thus the mechanism by which the critical surface tension controls the patency of a biomedical implant may not be that it prevents the formation of thrombus at all, but rather that it controls the adhesive events at the interface and allows the detachment of small emboli which can be coped with in the general blood stream. In Figure 7 we have summarized our over-all understanding as of this date of the interaction of blood with nonphysiologic surfaces. Although this figure is self explanatory, there can be disagreement with some of the specific hypothetical steps that are shown. The important point for the present presentation is that in each early step the surface energy factor is important. In particular, for the earliest obligatory steps, the adsorption of proteins and the adhesion of the saturated layer of platelets, the surface energy of the substrates seems to be the controlling factor. Although the surface energy of the substrates is a very difficult parameter to obtain, the critical surface tension (ye) can be obtained very handily in simple laboratory experiments and is directly relatable to surface energy. It is therefore confidently predicted that ye will continue to be used to diagnose these potential interVol. 48, No. 2, February 1972

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actions and eventually to dictate and control in advance the type of interaction that the blood will have at new prosthetic interfaces.
REFERENCES
1. Sawyer, P. N., editor: Biophysical Mechanisms icz Vascular Homeostasis and Intravascular Thrombosis. New York, Appleton Century Crofts, 1965. 2. Costello, M., Stanczewski, B., Viresman, P., Lucas, T., Srinivasan, S. and Sawyer, P. N.: Trans. Amer. Soc. Artif. Intern. Organs 16:1-6, 1970. 3. Baier, R. E., Gott, V. L. and Furuse, A.: Trans. Amer. Soc. Artif. Intern. Organs 16:50, 1970. 4. Rosenberg, N., Martinez, A., Sawyer, P. N., Wesolowski, S. A., Postlethwait, R. W. and Dillon, M. L.: Ann. Surg. 164:247, 1966. 5. Baier, R. E. and DePalma, V. A.: In: Management of Arterial Occlusive Disease, Dale, W. A., ed. Chicago, Yearbook Med. Publ., 1971. (i. Bokros, J. C. and LaGrange, L. D.: The Compatibility of Carbon and Blood. Contract Report No. GA-9777. San Diego, Calif., Gulf General Atomic Corp., 1969. 7. Zisman, W. A.: Relation of Equilibriwm, Contact Angle to Liquid and Solid Constitution. Advances in Chemistry Series, No. 43. Washington, D.C., Amer. Chem. Soc., 1964, p. 1. 8. Baier, R. E., Shafrin, E. G. and Zisman, W. A.: Science 162: 1360, 1968. 9. Harrison, J. H.: Amer. J. Surg. 95:3, 1958. 10. Hall, C. W., Spira, M., Gerow, F., Adams, L., Martin, E. and Baron Hardy, S.: Trans. Amer. Soc. Artif. Intern. Organs 16:12, 1970. 11. Lyman, D. J., Brash, J. L. Chaikin, S. W., Klein, K. G. and Carini, M.: Trans.
Amer. Soc. A rtif. Intern. Organs 14: 250, 1968. Dutton, R. C., Baier, R. E., Dedrick, R. L. and Bowman, R. E.: Trans. Amer. Soc. Artif. Intern. Organs 14:57, 1968. Friedman, L. I., Liem, H., Grabowski, E. F., Leonard, E. F. and McCord, C. W.: Trans. Amer. Soc. Artif. Intern. Organs 16:63, 1970. Petschek, H. E., Adamis, D. and Kantrowitz, A.: AVCO Everett Res. Lab. Report No. 314, 1968. Lyman, D. J., Muir, W. M. and Lee, I. J.: Trans. Amer. Soc. Artif. Intern. Organs 11:301, 1965. Baier, R. E., Dutton, R. C. and Gott, V. L.: In: Surface Chemistry of Biological Systems, Blank, M. editor. New York, Plenum, 1970. Taylor, A. C.: In: Biological Interactions in Normal and Neoplastic Growth, Brennan, M. J. and Simpson, W. L., editors. Boston, Little, Brown, 1962. Baier, R. E.: Chap. 2. In: Adhesion in Biological Systems, Manly, R. S., editor. New York, Acad. Press, 1970. Baier, R. E., DePalma, V. A., Gott, V. L. and Sawyer, P. N.: Unpublished studies. Dale, W. A. and Lewis, M. R.: Ann. Surg. 169:927, 1969. Baier, R. E. and Dutton, R. C.: J. Biomed. Mater. Res. 3:191, 1969. Dutton, R. C., Webber, T. J., Johnson, S. A. and Baier, R. E.: J. Biomed. Mater. Res. 3:13, 1969. Baier, R. E., Loeb, G. I. and Wallace, G. A.: Fed. Proc. 30:1523, 1971.

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