Fig. 1: Deadhesion of a thin soft patch adhering to a surface that transitions from smooth to sinusoidally-rough.

TopoGrafts: Synthetic grafts with long term patency for diabetic lower extremity bypass surgery

1. Topic area We have discovered that a change in surface topography (i.e. a smooth surface becomes rough or vice versa) can force deadhesion of adlayers that form on surfaces. Blood contact experiments show that synthetic tubular grafts whose inner surfaces undergo such changes in topography (dubbed TopoGrafts) show greatly reduced platelet adhesion. We will conduct translational research to develop TopoGrafts, primarily for treatment of diabetic vascular disease, and secondarily to address women’s heart disease.

Diabetes is more prevalent in the veteran population (~25%) compared to the general population(~8%)[1, 2]. Diabetic foot infection is a highly morbid complication of the disease due to resultant nonhealing wounds and progression to major foot or limb amputations. In recent years, VA has conducted 3500-4500 diabetes-related amputations per year[3, 4], and a recent article estimates the direct cost to the VHA healthcare system to be $200 million[2]. Limb amputation can be avoided by vascular surgery, and the VHA performs roughly 5000 vascular surgeries each year, of which well over half are proximal or distal bypass surgeries[5]. Such limb salvage is especially difficult because of vascular insufficiency, often in the form of small vessel or tibial disease that is common with long standing diabetes. Successful revascularization requires bypass to small caliber blood vessels using autologous veins as the ideal conduit [15-16]. Given the systemic nature of diabetes, these patients suffer from systemic vascular disease and often have undergone prior revascularization of other extremities as well as the heart [17,18]. Depletion of sources of suitable conduit is a frequent problem encountered by vascular and cardiac surgeons caring for diabetic patients. The use of prosthetic grafts in such vascular beds yields poor patency and limb salvage results [19]. In addition, when there is vein available, vein harvest through sizable incisions is especially morbid in diabetic patients who suffer from compromised wound healing. Our Topograft technology addresses the need for better graft materials that will have improved long term patency in smaller diameter targets such as the small tibial vessels often affected in diabetics.

While diabetic treatment is the primary target of our proposal, the principle of deadhesion by topographic actuation is generalizable. For instance, the exact same grafts developed in this project address a second area of interest to the PRMRP, women's heart disease. Surgeons perform nearly 500,000 coronary artery bypass grafting (CABG) operations annually (roughly 4000 in the VHA healthcare system[6, 7]), and in all cases the patients’ own veins or arteries must be harvested for the operation. This increases patient morbidity, and moreover may pose severe challenges if patients need re-operation, or if the most desirable blood vessels have already been used, e.g. for peripheral bypasses. Synthetic grafts would be far preferable to harvesting the patients’ autologous conduit since synthetic grafts reduce operative time and decrease the potential for wound healing complications at the harvest site. Unfortunately, synthetic grafts perform poorly small (<6 mm) caliber blood vessels such as the coronary arteries [20]. This is especially relevant for women’s heart disease due to their smaller vasculature. The development of off-the-shelf

synthetic vascular grafts that have improved patency in small vascular beds would dramatically improve outcomes for such patients.

In summary, TopoGrafts provide a novel self-renewing surface strategy to prevent platelet and thrombus adhesion. This approach is based on an entirely new mechanism based on mechanical aspects of adhesion rather than the chemical modification approaches that have had only a modest effect on long-outcomes. Topographic actuation can readily be combined with existing chemical modifications like heparin bonding for potentially synergistic effects, where the active topography keeps the chemically modified surface clean for better long term efficacy [21,22]. Synthetic small-diameter TopoGrafts are relevant to two areas of interest to the DoD:

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E

A B

C

D

low pressure

high pressure

lower extremity bypass surgery for diabetics, and CABG operations for women. In both cases, TopoGrafts promise a prosthetic alternative for autologous grafts with improved long term patency.

2. Technology/Therapeutic Development Product The principle of deadhesion by topographic actuation is illustrated in Fig. 1. A thin, soft film of adhesive material such as a thrombus, adheres to an initially-flat surface. The surface is then actuated (see below) so that it develops a rough (in our case sinusoidal) topography, which induces deadhesion of the film. We have developed an energy-based mechanics model of this process[8]. Briefly, the adhered film seeks to conform to the changing curvature of the surface, but this increases its elastic energy. Beyond a certain critical curvature, the elastic energy stored in the deformed film overcomes the adhesion energy, thus inducing deadhesion. This model predicts that the minimum roughness amplitude needed for deadhesion decreases with decreasing adhesion strength and decreasing wavelength of the roughness, in excellent agreement with finite element simulations[8].

Fig. 2: A. Bilayer of a stiff film attached to a soft substrate (top) wrinkles when compressed (bottom). An example of a stiff plastic film bonded to a soft rubber substrate is shown alongside[10]. B. Schematic of reversible inflation and deflation causing wrinkling inside a cylindrical tube whose inner surface layer is stiffer than the thicker outer layer. C. OCT image of the cross section of cylindrical silicone tube undergoing wrinkling over its luminal surface. The dotted blue line indicates the faintly visible outer surface of the tube. D. Macroscopic view of the cylindrical sample with end cut to show the two layered wall. E. Platelet adhesion on various grafts in pulsatile flow (see text).

Topographic actuation can be implemented by any suitable mechanism. One approach is the compression-induced wrinkling of a bilayer composite film comprising a thin stiff layer bonded a thicker soft layer (Fig. 2A)[9]. What is particularly convenient for our research is that micron-scale wrinkles develop spontaneously without needing to pattern the sample at micron-scales, and hence large (10 cm-scale) samples that wrinkle uniformly can be prepared. For the target application of blood vessels, the pulse pressure itself can be exploited to drive the compression-tension, and hence topographic actuation, of the grafts (Fig. 2B). Accordingly we constructed tubular grafts comprising a stiffer inner layer bonded to a thicker softer outer elastomeric tube (Fig. 2C,D), with the layers being composed of two different silicone rubbers. By judicious material selection, the tubes were designed to undergo 20% perimeter expansion from diastole to systole. Furthermore, by controlling strain at various stages of the fabrication, the tubes were designed to have a smooth lumen in the fully-stretched state (systole), whereas during the diastole, the stiff inner layer experienced compression and hence wrinkled (Fig. 2C). The wrinkle wavelengths could be tuned in the 50-1000 μm range simply by varying the thickness of the inner stiff

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layer.

Fig. 2E summarizes our in vitro experiments using a pulsatile blood pump. The two control samples, titanium (Ti) and silicone rubber grafts with a smooth lumen that are static i.e. do not alternate between smooth and wrinkled (Si). Both show high platelet adhesion. Samples I, II, and III correspond to grafts that continually expand and contract, with corresponding wrinkling-unwrinkling changes in lumen topography (verified by optical coherence tomography, e.g. Fig. 2D). The three wavelengths were 1000 (I), 250 (II) and 80 (III) μm respectively. All three showed sharply reduced platelet fouling, with the smallest wavelength corresponding to the least fouling. Indeed this is exactly in line with the theory and simulation that predicts that topographically-actuated deadhesion is more effective if the folds or wrinkles have a shorter wavelength.

These results suggest two approaches to improving long-term patency by decreasing platelet surface adhesion of synthetic grafts (1) the graft must continually expand and contract, analogous to the expansion/contraction of natural arteries during every pulse cycle, and (2) the graft must have an internal surface that continually transitions from smooth to rough at roughly few-10 μm lengthscales. It is critical to note that grafts used in current practice undergo negligible expansion-contraction under pulse pressure owing to their high stiffness. Moreover, while some of them happen to have a rough lumen (e.g. because they are woven fibers), static roughness does not suppress biofouling (indeed it may worsen biofouling slightly). Thus the proposed grafts are altogether different from current practice and if successful, will require a radical rethinking of the design of arterial grafts.

3. Research strategy Validating results in vivo: Although our preliminary findings show enormous promise, they were conducted in vitro using a pulsatile blood pump. The first goal of this research plan is to verify that TopoGrafts are efficacious in vivo. Specifically, we hypothesize that all-silicone tubular TopoGrafts will show lower biofouling in animal (pig) experiments as compared to control samples. We initially plan to perform non-survival 12 hour experiments where silicone TopoGraft (3 mm inner diameter) will be implanted as an interposition graft in the common carotid artery in pigs. Graft mechanics will be monitored with an OCT catheter and platelet adhesion evaluated using standard methods as in our in vitro work[8]. These initial experiments will provide important data on possible needed graft design optimization to take into account variations in cardiovascular hemodynamics. Once material optimization is performed (see below), we will conduct longer-term experiments that will observe graft behavior out to 8 weeks. The primary endpoint from this experiment will be graft patency, and the secondary endpoint will be surface platelet/thrombus adhesion.

TopoGrafts from FDA-approved materials: Our past research used materials that were optimized to test the basic hypothesis that topographic actuation reduces biofouling. These materials were not medical grade. In most current arterial grafts the lumen (i.e. the portion that is intended to contact blood) is one of just two materials: polyethylene terephthalate (PET, often referred to as Dacron®) and polytetrafluoroethylene (PTFE, often called Teflon®). We hypothesize that TopoGrafts made of medical grade silicone with a PTFE or PET lumen which continually wrinkles and unwrinkles will exhibit improved long term patency and overall decreased platelet adhesion. In effect, we will combine the known low adhesion of the current materials PTFE/PET with the idea of topographic actuation. We do anticipate two immediate technical challenges. First, PET and PTFE are difficult to cast into films; PTFE in particular is not soluble in any solvent at room temperature. Second, PET and PTFE are roughly 1000-fold stiffer than the silicone used as the inner layer of our previous TopoGrafts. The wrinkle wavelength of bilayers scales with the 1/3rd power of the modulus of the stiffer layer[11, 12], thus, realizing 10 μm -scale topography requires that the PET or PTFE thickness be almost 10-times smaller than our all-silicone grafts, roughly 0.5-1 μm.

We will tackle these challenges using a particle-deposition approach using PTFE (since micropowdered PTFE is available commercially). PTFE powder will be deposited on the outer surface of acrylic cylinders by dip coating or spraying to realize a micron-scale film. This will be dried, and then coated with a medical grade silicone (several commercial grades are approved for implantation). The remainder of the

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fabrication will be identical to that used in our previous research. In vitro and then animal implantation experiments will be conducted to test the hypothesis mentioned above. Similar experiments will be conducted with PET although in this case microparticles are not available; micropowders will be prepared using established methods using a high pressure solution precipitation[13].

Overcoming material constraints: new approach to topographic actuation: In our all-silicone TopoGrafts, as well as the PTFE or PET-based TopoGrafts proposed above, we exploit compression-induced buckling as the mechanism for topographic actuation. Although convenient (large wrinkled samples can be made without need for microfabrication or patterning), it imposes severe material constraints: we must pair a stiff material with a soft one, and furthermore the stiff material must be in the form of a thin film, and the topography (e.g. wavelength) depends on the mechanical properties. In the last part of the project, we seek to overcome these constraints with a patterning approach. Simulations suggest that by patterning the surface, e.g. with ridges or dimples, it can buckle readily, and this buckling can be tuned geometrically, independent of the mechanical properties, indeed even from a single material. Initial research in this direction will use standard microfabrication approaches to realize the desired patterns. After the geometric and material properties are selected, we will partner with a commercial extrusion processing company to realize these (more complex) grafts in a continuous extrusion process. In vitro (with a pulsatile pump) and animal experiments will be conducted at all stages.

4. Personnel This project is a collaboration between three investigators with complementary skills. Dr. Sachin Velankar is a Chemical Engineering faculty with expertise in experimental polymer science and wide-ranging knowledge of materials selection and processing of polymers. Dr. Edith Tzeng is an academic vascular surgeon with expertise in translational vascular research, large animal models, and clinical vascular reconstruction. She brings the knowledge of the shortcomings of current vascular conduits and the properties that would be desirable in novel constructs. Finally, Dr. Luka Pocivavsek is a resident in the Department of Surgery. Unusual for a surgeon, during his Ph.D. he developed enormous expertise with theoretical mechanics and numerical simulations of the same. He continues his research on complex mechanics problems of relevance to surgery. Dr. Pocivavsek has actually performed the experimental and numerical research thus far but will shortly resume his surgical training, and his past knowledge and experience is critical to continued progress.

5. Impact In vitro experiments strongly support the essential hypothesis that topological actuation reduces platelet adhesion and hence reduces biofouling in blood contact. The proposed research will have the following outcomes. (1) As a first step to translation into clinical practice, this grant will support animal experiments to evaluate the efficacy of topological actuation when actually implanted. (2) As a second step to translation, this grant will support experiments with grafts using FDA-approved materials which, due to their limited range of properties impose severe design constraints, and require new fabrication procedures. (3) As a third step to translation, we will develop TopoGrafts that are prepatterned to wrinkle at specific wavelengths and collaborate with a commercial manufacturer to scale up production by extrusion.

The immediate short term impact on the research field is to illustrate the importance of vascular compliance and dynamic topography in promoting deadhesion of platelets. At present, synthetic vascular grafts are made of stiff materials with negligible ability to expand or contract when implanted. Yet native blood vessels have a highly wrinkled lumen and expand and contract due to pulse pressure in normal blood flow[14]. We anticipate that this novel research will sharply revise the design considerations for synthetic grafts with paradigm shifting impact and provide a new generation of materials. Having off-the-shelf grafts for lower extremity and cardiac bypass readily available would tremendously impact the care of a large and growing patient population.

6. Military Relevance In the field, extremity injuries are numerous. Vascular reconstruction may be facilitated by reliable

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conduits that have superior patency and require less operative time to implant. In the longterm, veterans have an increased burden of diabetes and cardiovascular diseases. As a result, amputation rates are much higher in the veteran population. At present, there are no suitable synthetic grafts to help limb salvage of diabetic veterans suffering from ischemic wounds. TopoGrafts provide a radically-new approach to synthetic grafts that resist biofouling and will likely have superior long-term patency for lower extremity grafts. Furthermore, the technology is very general and hence will impact all small-diameter vascular surgery – especially relevant for women’s heart disease due to smaller vasculature.

References

1. U.S. Dept. of Veterans Affairs, Close to 25 percent of VA Patients Have Diabetes. Available from: http://www.va.gov/health/NewsFeatures/20111115a.asp.

2. Franklin, H., et al., Cost of lower-limb amputation in US veterans with diabetes using health ser-vices data in fiscal years 2004 and 2010. Journal of Rehabilitation Research and Development, 2014. 51(8): p. 1325-1330.

3. Sambamoorthi, U., et al., Initial nontraumatic lower-extremity amputations among veterans with diabetes. Medical Care, 2006. 44(8): p. 779-787.

4. Mayfield, J.A., et al., Trends in lower limb amputation in the Veterans Health Administration, 1989-1998. Journal of Rehabilitation Research and Development, 2000. 37(1): p. 23-30.

5. Mayfield, J.A., et al., Trends in peripheral vascular procedures in the Veterans Health Administra-tion, 1989-1998. Journal of Rehabilitation Research and Development, 2001. 38(3): p. 347-356.

6. Cornwell, L.D., et al., Changes Over Time in Risk Profiles of Patients Who Undergo Coronary Artery Bypass Graft Surgery The Veterans Affairs Surgical Quality Improvement Program (VASQIP). Jama Surgery, 2015. 150(4): p. 308-315.

7. Bakaeen, F.G., et al., Trends Over Time in the Relative Use and Associated Mortality of On-Pump and Off-Pump Coronary Artery Bypass Grafting in the Veterans Affairs System. Jama Surgery, 2013. 148(11): p. 1031-1036.

8. Pocivavsek, L., et al., Geometric tools for controlling soft surface adhesion. In preparation.9. Bowden, N., et al., Spontaneous formation of ordered structures in thin films of metals sup-

ported on an elastomeric polymer. Nature, 1998. 393(6681): p. 146-149.10. Pocivavsek, L., et al., Stress and fold localization in thin elastic membranes. Science, 2008.

320(5878): p. 912-916.11. Allen, H.G., Analysis and Design of Structural Sandwich Panels. 1969, New York: Pergammon.12. Huang, Z.Y., W. Hong, and Z. Suo, Nonlinear analyses of wrinkles in a film bonded to a compliant

substrate. Journal of the Mechanics and Physics of Solids, 2005. 53(9): p. 2101-2118.13. Tapriyal, D., et al., Micronization of polyethylene terephthalate via freezing of highly sheared

emulsions of polyethylene terephthalate in saturated liquid tetrahydrofuran. Journal of Applied Polymer Science, 2012. 125(5): p. 4034-4040.

14. Greensmith, J.E. and B.R. Duling, MORPHOLOGY OF THE CONSTRICTED ARTERIOLAR WALL - PHYSIOLOGICAL IMPLICATIONS. American Journal of Physiology, 1984. 247(5): p. H687-H698.

15. Mamode, N., Scott, R.N., “Graft type for femoro-popliteal bypass surgery”, Cochrane Database of Systematic Reviews, 2, CD001487 (1999, re-reviewed 2009)

16. Scharn, D.M. et al. “Biological Mechanisms Influencing Prosthetic Bypass Graft Patency: Possi-ble Targets for Modern Graft Design”, Eur. J. Vasc. and Endovasc. Surg., 43, 66-72 (2012)

17. Prompers L., et al. “Delivery of care to diabetic patients with foot ulcers in daily practice: results of the Eurodiale Study, a prospective cohort study. Diabet. Med. 25:700-707 (2008).

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18. Prompers, L. et al. “High prevalence of ischemia, infection and serious comorbidity in patients with diabetic foot disease in Europe. Baseline results from Eurodiale study. Diabetologia. 50: 18-25 (2007).

19. Albers, M., et al., “Meta-analysis of alternate autologous vein bypass grafts to infrapopliteal ar-teries.” J. Vasc. Surg. 42: 449-455 (2005).

20. Desai, M. et al. “Role of prosthetic conduits in coronary artery bypass grafting”, European Jour-nal of Cardiothoracic Surgery, 40, 394-298 (2011)

21. Devine, C. and McCollum, C., “Heparin-bonded Dacron or PTFE for femoropoliteal bypass: five-year results of a prospective randomized multicenter clinical trial”, J. Vasc. Surg. 40(5), 924-931 (2004).

22. Ye SH, Johnson CA Jr, Woolley JR, Murata H, Gamble LJ, Ishihara K, Wagner WR, “Simple surface modification of a titanium alloy with silanated zwitterionic phosphorylcholine or sulfobe-taine modifiers to reduce thrombogenicity”, Colloids and Surfaces B: Biointerfaces 79:357-64 (2010).

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