The "Theta Surface" Alternative to Animal Testing

There is a universal surface quality that can be imparted to any material, prior to its exposure to any biological system, that will least denature—change the conformation of—protein-based macromolecules that inevitably deposit and attach to it. Associated with this surface state is the ability to easily shed accumulating biomass in the same fashion that killer whales and dolphins (Figure 1) remain free of biofouling over lifetimes in seas of bioadhesive organisms. This quality is defined as the “theta surface”, by analogy with the “theta solvent” concept for solution-state macromolecules introduced by Flory (1), and in recognition of historical use of the “theta” symbol for contact angle values (2) upon which this new concept is based. As a consolidation of findings from over 40 years of laboratory, animal, and clinical research and testing, this concept can lessen the costs and consequences of animal use (3) as an evidence-based biomaterials selection criterion. In essence, we can copy properties of Nature onto materials of polymeric, metallic, and ceramic fabrication to attain (or resist) biomass easy-release outcomes for mechanical forces available in synthetic blood flow circuits and lab-on-a-chip devices.

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Figure 1. The natural surface properties of killer whales and dolphins easily shed depositing biofouling, sharing these properties with the similar natural “easy-release” surfaces of human oral mucosa and blood vessel intima.

In 1959, as a surgical technician, I constructed and operated artificial kidneys and heart-lung machines on large test animals as well as early human patients. Most died. One of my duties was to spray-clean, with boiling lye solution, the heart-lung machine parts between surgeries, to eliminate tenaciously attached proteinaceous and thrombotic deposits from all the blood-contacted components. This began my lifelong curiosity about how one might minimize protein adhesion to bioengineering materials. For 20 years, beginning in 1964, the Artificial Heart Program of the U.S. National Institutes of Health supported industry/university/contractor teams engaged in animal implantation studies of the commodity materials now dominant in the 200 or so different implantable parts surgically placed into human hosts, with reasonable success. Unfortunately, the NIH investment plan that led to these “biocompatible” materials selections was curtailed (4), and a “biomaterials availability” crisis emerged as industry owners of commodity materials withdrew them from medical marketplaces in response to increasing litigation costs (5). New materials still are sought!

In addition to minimizing animal sacrifice while serving an urgent need for discovery-based selection of materials that resist biofouling in medical and dental restorative and therapeutic devices, in food and pharmaceutical processing, and for nontoxic, nonpolluting coatings of vessels in maritime commerce, “theta surface” selection should overcome fouling occurring in microfluidic circuits and biosensors (6).

Premises of the "Theta Surface" Concept

The main approaches to “biocompatibility” for over thirty years have been control of (a) surface charge, (b) surface texture, and (c) surface energy. Judge each concept by the practical products that have resulted and continue to benefit personal, public and environmental health; ask each concept’s proponents: “where are your successful products?” Here is the case for surface energy control—via simple contact angle measurements in accord with a strict protocol—as the dominant factor in modulating biological responses to synthetic materials.

Safe and effective, long-term biological responses are obtained to so many different materials, correlated with and controllable by surface energetic factors, that it is appropriate to consider this a “universal” approach to controlling all underwater interactions: witness the blood compatibility of Starr-Edwards heart valves [over 30 years], Dardik Biografts for limb salvage [15 years], pyrolytic carbon heart valves [over 15 million human patients], and the growing successes of the “Hershey heart” as a bridge to cardiac transplantation—and at least 9 similarly surface-energy controlled ship bottom paints to resist biofouling (7) now in the commercial marketplace based on the same concepts and polymers as used in artificial heart development. A successful correlating curve for these developments was first published to the Marine Technology industries in 1973 (8), and has become a generally accepted principle for new biofouling-resistant marine coatings (9). Figure 2 is a simplified version of this correlating curve, showing the “theta surface” quality is that obtained for materials exhibiting measured Critical Surface Tension (surrogate for theoretical surface free energy) values between 20 and 30 mN/m (mJ/m2).

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Figure 2. This is a “universal” summary plot correlating the relative underwater strengths of retention of all biological substances to all materials, with the Critical Surface Tension determined from empirical contact angle measurements using many test liquids. Note that the surface properties for most natural and synthetic “easy-release” surfaces fall into region III, while load-bearing dental implants require surface qualities associated with region II. Most commercial materials have surfaces characterized by region I, and give variable results in contact with biological substances. Minimizing biofouling within microfluidic circuits or biosensor devices would result from conversion of device material surface properties to those of region III.

With Critical Surface Tension values on the rising slope (regions I to II) in Figure 2, secure biological adhesion is routinely obtained to polyethyleneterephthalate vascular grafts and commercially pure titanium dental implants, many millions implanted in people around the globe for more than 3 decades. These utilitarian results have emerged from 3 decades of concurrent inquiry into Nature’s own material surface properties: natural skin surfaces, cartilage, and teeth have higher surface energies and strong bioadhesion, while interior walls of blood vessels, the eye’s cornea, red blood cell surfaces, intra-oral mucosa, temporomandibular discs, porpoise and killer whale integuments, canine heartworms, gorgonian corals, agar and confluent lawns of bacteria all exhibit “theta surface” easy-release properties.

Experimental and Theoretical Aids to Material Selection

Here is a brief overview of the path recommended for an empirically sound and theoretically reasonable approach to prediction and beneficial control of biological responses to nonphysiologic materials by modulation of the surface energetics of the components interacting under water. It is axiomatic that actual interactions of materials in biological settings require that water be displaced from the interface—so measurements of aqueous contact angles are useful mainly to estimate how long it will take before the important material-to-biopolymer contacts will occur. Water contact angle data, alone, are not sufficient to determine or correlate bioadhesive strengths developed when—inevitably—interfacial dehydration takes place.

As an example, note that soft contact lenses—some with more than 70% initial water content—do always become severely soiled by proteinaceous matter from the tears. There are no synthetic hydrophilic or hydrogel coatings surviving unfouled for a year by organisms in the sea!

Beyond hydrophilicity, the complete range of wetting, spreading and adhesive interactions important to understanding, predicting and controlling biosurfaces can be easily obtained, however, by extending the measurements of contact angle values to include representative pure liquids for each of the multiple side chains of protein-building amino acids. Relative water wettabilities of materials are certainly not predictive, alone, of the surface energetics of biomaterials. Although experiments that take only minutes to weeks are not adequate, alone, to confirm or refute the predicted long-term bioadhesive outcomes critical to successful medical implants or ship bottom paints, sufficient clinical data in human patients and actual seawater environments are now available to support direct transfers from laboratory to practice without needing to sacrifice living species on the way.

Differential adhesion in all biological systems is strongly correlated with substratum surface energy (10), transduced to the level of particulate matter—living or dead—via universally deposited and preferentially retained proteinaceous “conditioning” films (11)—that produce a nonlinear surface energy vs bioadhesion relation minimized at the 20-30 mN/m substratum region of the Critical Surface Tension scale (12). This is the domain of the “theta surface”!

Within any given biological system, there is a dominant identity of the proteins that deposit and are preferentially retained on all substrata, but these compositionally similar protein deposits do have different surface-energy-related conformations, which do also change through time (13). Within any given biological system, specific particles or cells dominate the “primary” particulate deposits onto the “conditioning” films, but these particles also show surface-energy-related differences in patterns and degrees of spreading, determined after contact with the pre-deposited “conditioning” films and not in the suspension state before surface contact (14). There is NO selectivity in adsorption of macromolecules or bacteria or cells on substrata in biological systems; rather there IS SELECTIVITY in retention against differential detachment forces as a function of the differing surface energetics associated with the initial concentration- and flux-driven deposition events (15). Therefore, it is essential that controllable mechanical work, such as shear stress, be present if relative bioadhesive strengths are to be reliably inferred (16).

Differential “processibility” of the deposited “conditioning” and “primary” layers by shear forces and local biochemical/cellular reactions determines whether the immersed substrata will be retained with their integral “biofilms” or will be “walled off” or dehisced in the classical “foreign-body” reaction (17).

As Figure 2 illustrates, there is NO finding of ZERO strength of retention of biomass to any underwater substratum. The absolute adhesion strengths vary with degree of surface polarity, time in contact, type of biology attached, and metabolic activities of the organisms.

Why the universal minimum in biological “stick-to-it-iveness” at about 22 mN/m Critical Surface Tension? The critical surface tension for spreading on a liquid substrate is 22 mN/m for interfacial water layers (18). Again, this is the domain of the “theta surface”!

Noting that this value also is equal to the dispersive force contribution to the composite surface free energy of water, an independently formulated explanation for the occurrence of a bioadhesion minimum on 20-30 mN/m low energy surfaces is that excess dispersion forces emanate from the solid surface on the high critical-surface-tension side of the minimum while they emanate from the liquid surface on the low critical-surface-tension side (19). The remainder of the theoretical argument follows the same logic as used in definition of “theta solvent”, for volumes that retain suspended polymers in their most ideally stable conformations.

With these experimental and theoretical findings now in place, Figure 3 illustrates the call to convert animal testing procedures to bench-level flow cell (20) studies in pursuit of new materials for use in biomedical implants and biosensor devices.

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Figure 3. This drawing illustrates the recommended transition from living animal testing for biocompatibility to bench-level testing with simple flow cells containing materials to be inspected by entirely in vitro criteria prior to acceptance for human use.

References

1. P.J. Flory, Principles of Polymer Chemistry, Cornell University Press, Ithaca, NY 1953
2. W.A. Zisman, Relation of the Equilibrium Contact Angle to Liquid and Solid Constitution, Advances in Chemistry 43: 1-51, 1964
3. A. M. Goldberg, T. Hartung, Alternatives to Animal Testing: The New Toxicology—Evidence Based Medicine Goes In Vitro, Scientific American, January 2006
4. R. E. Baier, The Future of Biomedical Implants, Pharmaceutical News 5: 25-29, 1998
5. J.A. Gould, B. Liebler, R. Baier, J. Benson, J. Boretos,T. Callahan, E. Canty, R. Compton, D. Marlowe, R. O’Holla, B. Page, J. Paulson, C. Swanson, Biomaterials Availability: Development of a Characterization Strategy for Interchanging Silicone Polymers in Implantable Medical Devices, Journal of Applied Biomaterials 4: 355-358, 1993
6. R.Mukhopadhyay, When Microfluidic Devices Go Bad, Analytical Chemistry, 429A- 432A, Nov 1, 2005
7. A.W. Wells, A.E. Meyer, J.A. Matousek, R.E. Baier, and E.F. Neuhauser, Nontoxic Foul-Release Coatings for Zebra Mussel Control. Waterpower’97: Proceeding of the International Conference on Hydropower, American Society of Civil Engineers, 1: 451-460, 1997
8. D.W. Goupil, V.A. DePalma, and R.E. Baier, Prospects for Nontoxic Fouling-Resistant Paints, Proceedings, Marine Industries: Problems & Opportunities, 9 Annual Conference, Marine Technology Society, Washington, DC, pages 445-458, 1973
9. R. F. Brady, Jr, I.L. Singer, Mechanical Factors Favoring Release from Fouling Release Coatings, Biofouling 15, 73-81, 2000
10. R.E. Baier, E.G. Shafrin, and W.A. Zisman, Adhesion: Mechanisms that assist or impede it., Science 162: 1360-1368, 1968
11. R.E. Baier and R.C. Dutton, Initial Events in Interactions of Blood with a Foreign Surface, Journal of Biomedical Materials Research 3: 191-206, 1969
12. R.E. Baier, Surface Properties Influencing Biological Adhesion, Chapter 2 in Adhesion in Biological Systems, R.S. Manley, Editor, Academic Press, New York, pages 15-48, 1970
13. R.E. Baier, G.I. Loeb and G.T. Wallace, Role of an Artificial Boundary in Modifying Blood Proteins, Federation Proceedings 30: 1523-1538, 1971
14. R.E. Baier, The Organization of Blood Near Interfaces, Annals of the New York Academy of Sciences 283: 17- 36, 1977
15. R.E. Baier, Conditioning Surfaces to Suit the Biomedical Environment: Recent Progress, Journal of Biomechanical Engineering, 104: 257- 271, 1982
16. R.E. Baier, A.E. Meyer, V.A. DePalma, R.W. King, and M.S. Fornalik, Surface Microfouling During the Induction Period, Journal of Heat Transfer 105: 618-624, 1983
17. R.E. Baier, A.E. Meyer, J.R. Natiella, R.R. Natiella, and J.M. Carter, Surface Properties Determine Bioadhesive Outcomes: Methods and Results, Journal of Biomedical Materials Research 18: 337-355, 1984
18. E.G. Shafrin and W.A. Zisman, Critical Surface Tension for Spreading on a Liquid Substrate, Journal of Physical Chemistry 71: 1309-1316, 1967
19. M.E. Schrader, On Adhesion of Biological Substances to Low Energy Solid Surfaces, Journal of Colloid and Interface Science 88: 296-297, 1982
20. R.E. Baier, A.E. Meyer, R.W. King, Improved Flow-cell Techniques for Assessing Marine Microfouling and Corrosion, in Marine Biodeterioration, (M.F. Thompson, R. Sarojini, R. Nagabushhanam, editors), Oxford & IBH Publishing Co., New Delhi, 1988