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Ann Thorac Surg 2006;81:47-55
© 2006 The Society of Thoracic Surgeons

Original article: Cardiovascular

Cholesterol-Modified Polyurethane Valve Cusps Demonstrate Blood Outgrowth Endothelial Cell Adhesion Post-Seeding In Vitro and In Vivo

Stanley J. Stachelek, PhD a , Ivan Alferiev, PhD a , Jeanne M. Connolly, MS a , Michael Sacks, PhD b , Robert P. Hebbel, MD c , Richard Bianco, BA d , Robert J. Levy, MD a , *

a Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
b McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
c Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota
d Department of Surgery, University of Minnesota School of Medicine, Minneapolis, Minnesota

Accepted for publication July 18, 2005.

* Address correspondence to Dr Levy, The Children's Hospital of Philadelphia, 3615 Civic Center Blvd, Abramson Research Center, Suite 702, Philadelphia, PA 19104-4318 (Email: levyr{at}email.chop.edu).


Mr Bianco discloses a financial relationship with Medtronic.

 

   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: The clinical and experimental use of polyurethane heart valve prostheses has been compromised by thrombosis and calcified thrombus. This is caused in part by the lack of an intact endothelium on these implant surfaces. We hypothesize that endothelial seeding of a polyurethane heart valve leaflet with autologous sheep blood outgrowth endothelial cells (BOECs) could be achieved with cholesterol-modified polyurethane (PU-Chol) to promote BOEC adhesion, thereby resulting in an intact, shear-resistant endothelium that would promote resistance to thrombosis.

METHODS: Cholesterol-derivatized polyurethane was formulated by bromoalkylation of the urethane nitrogens followed by reactive attachment of mercaptocholesterol. In vitro shear flow studies were carried out comparing BOEC retention on control surfaces versus PU-Chol using forces comparable to those observed in vivo with cardiac valves (75 dyne/cm2). Autologous sheep BOECs were seeded onto PU-Chol before pulmonary leaflet replacement surgery under cardiopulmonary bypass. Studies were terminated at 30 and 90 days followed by retrieval analyses.

RESULTS: Blood outgrowth endothelial cell seeding of PU-Chol surfaces resulted in an endothelial monolayer that was positive for von Willebrand factor. Polyurethane-cholesterol demonstrated significantly greater BOEC adhesion under 75 dyne/cm2 shear force in vitro than control polyurethane (75.3% ± 12.3% versus 5.8% ± 3.9%, respectively; p < 0.001). Sheep pulmonary cusp replacements demonstrated retention of seeded BOECs on PU-Chol leaflets with no significant differences in the extent of cellular density comparing unimplanted specimens with explants. Control explants (nonseeded PU-Chol and nonseeded polyurethane) demonstrated no evidence of endothelial recruitment.

CONCLUSIONS: Polyurethane-cholesterol represents a polyurethane formulation with very high adhesive properties for BOECs under heart valve level shear forces both in vitro and in vivo.

Polyurethane (PU) elastomers have been used both clinically [1–3] and experimentally [4, 5] in left ventricular assist systems and total artificial heart implants as a key component material for flexing, blood-contacting surfaces. However, PU heart valve prostheses, typically in trileaflet valve designs, have been investigated to a very limited extent clinically and more extensively in experimental settings, in which cuspal dysfunction leading to device failure has frequently occurred as a result of both thrombosis and calcification [6–9]. Polyurethane cuspal calcification occurs on the blood-contacting surfaces of these devices, and in general results from calcified thrombus [10]. This is caused in part by the lack of an intact endothelium on these PU implant surfaces, with resulting thromboembolic activity.

Our group has recently demonstrated that mercaptocholesterol could be reactively attached to bromoalkyl-activated PU, thereby rendering the PU surface highly lipophilic and endothelial-adhesive with significantly greater retention of both mature endothelial cells and blood outgrowth endothelial cells (BOECs) in vitro under simulated arterial shear (25 dyne/cm2) stress conditions [11]. Blood outgrowth endothelial cells are an outgrowth of a circulating progenitor cell, present in peripheral blood, and thus represent an important potential source of autologous cells for seeding investigations [12, 13]. The present studies investigated the hypothesis that cholesterol-modified polyurethane (PU-Chol) will have increased adhesion of BOECs compared with controls under valvular level shear forces, 75 dyne/cm2 [14] in vitro, and thus will also support retention of BOEC seeding in vivo with PU-Chol pulmonary cusp replacements in sheep [12, 13].

Thus, the goals of these investigations were to investigate this hypothesis by formulating PU-Chol and investigating the retention of sheep BOECs on the surface of PU-Chol under both simulated heart valve shear force conditions in vitro and in vivo using autologous BOEC-seeded PU-Chol pulmonary valve leaflet replacements in sheep.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Polyurethane and Polyurethane-Cholesterol Synthesis
The PU used in these studies, Tecothane TT1074A (Thermedics, Waltham, MA), a polyether polyurethane, was used either unmodified in dimethylacetamide solution, or covalently modified with cholesterol (Chol) by means of bromoalkylation to form PU-Chol as previously published (11) and then dissolved in dimethylacetamide. Polyurethane and PU-Chol dissolved in dimethylacetamide were solvent cast as films as previously described [11]. These films were routinely between 159 and 220 µm thick [11], and were cut to size as needed for biomechanical studies, insertion into cell culture wells (1 x 1 cm) or formation of leaflets for sheep pulmonary valve replacement (3 x 3 cm). Alternatively, for shear adhesion studies, films were cast directly onto glass slides [11].

Biaxial Stress–Strain Studies of Polyurethane-Cholesterol and Polyurethane
Sample films were loaded into a Tytron 250 (MTS Systems Corp, Eden Prairie, MN) axial-loading test frame. The samples were subjected to uniaxial tension with a strain rate of 0.1 mm/s to a maximum strain of 200%. The samples were not taken to failure because their deformation exceeded the limits of the device used. Once the tensile test was finished, the force–displacement data was used to generate stress–strain curves. A sixth-order polynomial was fit to all the stress–strain curves (r 2 > 0.99), and the first derivative was taken to yield the instantaneous slope of the curve extrapolating to 0% strain to obtain a characteristic material modulus value.

Cells and Cell Culture
Sheep BOECs were isolated from study sheep with freshly drawn peripheral blood as previously reported [11, 12] in accordance with an approved Institutional Animal Care and Use Committee protocol at the University of Minnesota, and maintained in EGM-2 medium (Cambrex, East Rutherford, NJ). Cells isolated using this protocol express endothelial markers, including von Willebrand's factor (VWF), Flk-1, P1H12, and VE-cadherin [12, 13, 15] and demonstrate uptake of acetylated low-density lipoprotein. Blood outgrowth endothelial cells were used between passages two and ten, and were further characterized by Western blots (see below) before initiation of this study.

Blood outgrowth endothelial cells were routinely seeded at a density of 0.5 x 106 cells per square centimeter. Polyurethane-cholesterol leaflet implants that were previously seeded with cells were maintained submerged but slightly above the surface of a Petri plate by means of wound clips attached to the corners of the material, or when destined for surgical implantation, by clamping into a customized leaflet holder device (see below and Fig 1). Each side of the implant was then seeded as above, but with a 5-hour incubation between sides to allow the BOECs on the first side to adhere before inversion for plating the second side. Cells reached 80% to 100% confluence in 4 days after plating (data not shown), and were used at confluence for all studies unless otherwise noted.



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  		Fig 1. Implantation of cell-seeded polyurethane valve leaflets. (A) Valve leaflet mounted in holder (arrow), which is used for manipulation both during cell culture and surgery. (B) Holder and trimmed leaflet shown with handle in place. (C) Sutures prepared on valve sewing annulus while still in holder, resulting in (D) implantation with minimal handling of seeded valve.

 
Collagen Synthesis
Tritiated-proline incorporation into cellular and extracellular matrix collagen was quantitated using the method of Murakami and associates [16]. Briefly, BOECs were plated in 24-well plates on a surface of either PU or PU-Chol. At approximately 90% confluence, cultures were washed and medium was replaced with fresh medium containing 10 µCi/mL 3H-proline (Perkin-Elmer, Inc, Boston, MA), and the incubation was continued for 24 hours. Wells were then washed with cold medium and digested with 125 U/mL Collagenase I (Sigma Chemical Co, St. Louis, MO) for 2 hours at 37°C. The resulting supernatant was removed, and the trichloroacetic acid-precipitable portion was resolubilized and counted on an LKB scintillation counter (LKB/Perkin-Elmer, Shelton, CT) with a beta counting efficiency of 40%. Results are presented as counts per minute per well, and the experiment was performed in triplicate.

Shear Adhesion Studies
FlexCell BOECs were grown to confluence on FlexCell-specific culture slips (FlexCell International Corporation, Hillsborough, NC; 75 x 25 x 1 mm) coated with PU (with and without cholesterol modification). Uncoated culture slips, incubated with 2 mol/L NaOH for 1 hour, were used as a control substrate [11]. Up to six slides were placed in a parallel-plate flow system (Streamer; FlexCell International Corporation), which regulated the peristaltic pump-delivered flow of 37°C growth media supplemented with 5% fetal bovine serum and simulating aortic shear (75 dyne/cm2) over the cell monolayer. At the 2-hour cessation of the protocol, slides were washed with phosphate-buffered saline solution, and adhered cells were fixed in 4% paraformaldehyde and stained with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Retention was assessed by counting DAPI-positive cells in a minimum of nine random x200 fields.

Western Blot Analysis
Confluent BOECs, grown on 100-mm tissue culture–treated dishes, were washed with phosphate-buffered saline solution and scraped in ice-cold lysis buffer containing Tris-Hcl, 50 mmol/L, pH 7.4; 1% NP-40, sodium deoxycholate, 0.25%; NaCl, 150 mmol/L; EDTA, 1 mmol/L; PMSF, 1 mmol/L; aprotinin, 1 µg/mL; leupeptin, 1 µg/mL; pepstatin, 1 µg/mL; Na3VO4, 1 mmol/L; and sodium fluoride, 1 mmol/L. Cellular lysates were passed through a 21-gauge needle, and the proteins (25 µg) were reduced, denatured, and resolved on a 4% to 15% sodium dodecylsulfate–polyacrylamide gel electrophoresis gel according to the method of Laemmli [17]. Resolved proteins were transferred to a nitrocellulose membrane for immunoblotting. The antibodies used and concentrations were as follows: rabbit anti-CD31 (PECAM-1; Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:200 in Tris-buffered saline with 0.05% Tween 20 [TTBS] and 5% milk), rabbit anti-Flk-1 (Santa Cruz Biotechnology; diluted 1:200), and goat anti-Tie 1 (R & D Systems, Minneapolis, MN; diluted 1:500). Immune complexes were detected with the species-appropriate, horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) diluted 1:1000 in TTBS and 5% milk and the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

Sheep Study Design
Animals were assigned, in accordance with an approved Institutional Animal Care and Use Committee protocol at the University of Minnesota in compliance with the 1996 National Research Council Guide for the Care and Use of Laboratory Animals (http://www.nap.edu), to one of three groups: PU pulmonary valve leaflet replacement, PU-Chol valve leaflet replacement, and BOEC-seeded PU-Chol valve leaflet replacement. For BOEC-seeded PU-Chol films, sheep were entered into the study after successful isolation and expansion of autologous BOECs, which were used on seeded films. Polyurethane-cholesterol films were mounted in a holder (Fig 1) for facilitating seeding in cell culture and ultimately surgery. Duplicate seeded film preparations were transported to the surgical suite and manipulated in the same manner as were the pulmonary valve leaflet replacements, but removed from the surgical suite and fixed in 10% neutral buffered formalin for evaluation of cell loss as a result of the implantation procedure (handling controls). These handling controls were grossly evaluated and photographed and fixed in neutral buffered formalin at the University of Minnesota, then shipped to the Children's Hospital of Philadelphia for further study. Sheep in the PU and BOEC-seeded PU-Chol groups were subdivided into 30-day and 90-day implant duration groups.

Sheep Studies: Pulmonary Valve Leaflet Replacement and Retrieval
Pulmonary valve leaflet replacement surgery was performed on Dorset or Columbian crossbred female or castrated male sheep (4 to 5 months of age; Holly Neaton Farms, Watertown, MN) as previously described [11, 18]. Briefly, pulmonary valve leaflets were configured from PU, PU-Chol, or BOEC-seeded PU-Chol films with appropriate trimming in the operating room and, under general anesthesia and cardiopulmonary bypass, sewn in to replace excised native pulmonary valve leaflets. Handling of seeded implants was minimized by use of a customized holder (Fig 1) for the convenient and sterile transfer of the seeded leaflets to surgery. The holder assembly plus a handle allowed the surgeon to trim the sewing annulus edge in an arc suitable for the exact implant site and place sutures without significantly affecting the seeded cells (see below). Animals recovered from surgery and were allowed monitored pasture conditions for 30 or 90 days as designated by the study design. At specific times, animals were returned to the surgical suite, evaluated under general anesthesia, and sacrificed, and the pulmonary valve apparatus was retrieved intact for examination. After gross pathologic evaluation and photography, valve assemblies were fixed in 10% neutral buffered formalin and shipped to the Children's Hospital of Philadelphia for cell density determination and immunostaining (see below).

Polyurethane Leaflet Retrievals: Cell Density Determination and Immunostaining
Blood outgrowth endothelial cells seeded on PU-Chol, native sheep pulmonary valves, explanted pulmonary valve replacements, and handling controls were fixed in 10% neutral buffer formalin. Materials were stained either as floating mounts en face, or were frozen and stained as cross sections as described below. Gross observations were made of explants before dissection of valve leaflets, which were then mounted en face with DAPI to visualize nuclei. Using a Nikon Eclipse TE300 inverted fluorescent microscope (Nikon, Tokyo, Japan), the number of nuclei per 200x field were counted in approximately 10 fields per sample. Handling controls were treated in a like manner. Portions of each PU leaflet were divided and stained directly as floating mounts en face, and separately embedded as frozen sections in O.C.T. compound (Sakura Finetek USA Inc, Torrance, CA), and PU or PU-Chol explants were sectioned at 30 µm for immunostaining. In preliminary studies of seeded materials, this thickness was determined to be optimum for maintaining uniform cellular adhesion (data not shown). Sections were stained for von Willebrand's factor using a rabbit anti-human antibody (A0082, 1:500; DAKO, Carpinteria, CA) or for CD41/CD61 using a monoclonal anti-sheep antibody (Research Diagnostics, Inc, Flanders, NJ; 1:10), and both were developed using a Vector ABC secondary kit with DAB chromogenic development (DAKO). Hematoxylin and eosin stains (DAKO) were performed according to established procedure.

Statistical Analysis
Data are reported as mean ± standard error of the mean except as indicated. Statistical comparisons between groups were made using Student's t test using SigmaStat (version 3.0, SPSS, Inc, Chicago, IL) analysis software. Probability values equal to 0.05 or less were considered significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Cholesterol-Modified Polyurethane: Biomechanical Results
Uniaxial stress–strain experiments revealed no significant differences in elastic behaviors in comparisons between PU and PU-Chol (Table 1, Fig 2). The data indicate that between 50% and 200% deformation there were no significant differences noted in the force required to achieve these uniaxial dimensional changes. These deformations are far beyond those that would occur with heart valve leaflet biomechanics under physiologic conditions, and thus indicate that the cholesterol modification does not alter the essential elastomeric behaviors of Tecothane. Furthermore, the intrinsic elastic modulus (see Table 1, 0% deformation data), although somewhat lower for PU-Chol than PU, was not significantly lower. Taken together, these results demonstrate that PU-Chol should hypothetically perform comparably to PU in vivo.


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  		Table 1. Mean Elastic Modules Values for Given Strains: Comparisons with Unmodified Tecothane and Cholesterol Modified Tecothane
 


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  		Fig 2. Biomechanics: stress–strain behavior comparing cholesterol-modified polyurethane (two lower gray lines) with unmodified polyurethane (two upper black lines) films, demonstrating comparable elastic properties as indicated in plots of the elastic modulus versus applied strain.

 
Blood Outgrowth Endothelial Cells Cultivated on Polyurethane-Cholesterol: Characterization, Collagen Biosynthesis, and Shear Results
Ovine BOECs were expanded from peripheral blood-derived mononuclear cells. These cells were positive for the following characteristic endothelial markers: PECAM-1, Tie1, and Flk-1 per Western blot electrophoresis studies (Fig 3A). When cultivated on PU-Chol films, BOECs formed a monolayer of cells with a characteristic cobblestone appearance (Fig 3B); en face immunostaining revealed virtually all of the cells in the BOEC monolayer to be positive for von Willenbrand factor (Fig 3C), thus further documenting a persistent endothelial phenotype. Blood outgrowth endothelial cells were cultivated on PU and PU-Chol in the presence of 3H-proline to ascertain relative differences in collagen biosynthesis. Blood outgrowth endothelial cells cultivated on PU-Chol incorporated significantly greater amounts of 3H-proline than did BOECs grown on PU (Fig 4), thus indicating that collagen biosynthesis and very likely other extracellular matrix proteins are upregulated in BOECs grown on PU-Chol. This may contribute to the increased adhesion under flow conditions observed in vitro and in vivo (see below).



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  		Fig 3. Characterization of sheep blood outgrowth endothelial cells in vitro. (A) Western blot showing the presence of PECAM-1, Flk-1, and Tie1 proteins. (MW = molecular weight.) (B) Cobblestone morphology of blood outgrowth endothelial cells grown on polyurethane-cholesterol (hematoxylin and eosin stain, magnification x200). (C) Immunohistochemistry demonstrating von Willebrand's factor (brown stain; magnification x200), compared with (D) negative immunoglobin G control slide.

 


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  		Fig 4. Increased collagen synthesis by blood outgrowth endothelial cells grown on polyurethane-cholesterol (PU-Chol) compared with blood outgrowth endothelial cells grown on unmodified polyurethane (PU). *p = 0.006. (cpm = counts per minute.)

 
For shear-adhesion studies, BOECs were cultivated on glass microscope slides coated with either PU or PU-Chol as previously described [11]. Blood outgrowth endothelial cells were also cultivated on glass slides pretreated with sodium hydroxide as a positive high-adhesion control [11]. Tissue culture media was streamed over the surface of the seeded slides, and the extent of cell adhesion was documented by counting DAPI-stained nuclei per fluorescent microscopy. Studies compared cell retention using a constant fluid stream at valve shear force levels over the BOECs seeded on the test surfaces. After 2 hours simulating heart valve level shear force of 75 dyne/cm2, there was significantly greater BOEC retention on PU-Chol compared with controls under these high shear conditions (Fig 5A; p = 0.006). Blood outgrowth endothelial cells were more extensively retained on the surface of PU-Chol as noted by phase microscopy (Fig 5Bf) in contrast to unmodified PU, which showed widespread loss of seeded BOECs with time (Fig 5Bd).



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  		Fig 5. Adhesion of blood outgrowth endothelial cells exposed to constant flow. (A) Graphical representation of blood outgrowth endothelial cells retention after a 2-hour exposure to simulated valvular levels of shear flow (75 dynes/cm2). *p = 0.006. (B) Phase-contrast photomicrographs comparing representative starting fields of cells versus cells retained after 2 hours of shear flow: (a–c) time zero (t0); (d–f) 2 hours after flow (t2h) for blood outgrowth endothelial cells grown on polyurethane (PU: a, d), glass (b, e), and polyurethane-cholesterol (PU-Chol: c, f). Magnification x200.

 
In Vivo Experiments: Sheep Circulatory Results With Polyurethane Pulmonary Valve Leaflet Replacements
Polyurethane-cholesterol films (see above) were seeded on both sides with 5 x 105 cells/cm2 autologous BOECs on each side of the film 72 to 96 hours before open heart surgery for each individual animal. Seeded PU-Chol films were brought to the operating room in a 37°C transport incubator at the time of the cardiac procedure. Control implants were either nonseeded PU-Chol or nonseeded PU. Six animals died within 24 hours of surgery of surgical complications. No other early deaths occurred, leaving the following number of sheep completing each study arm: PU valve replacement, 8 (6 for 30 days and 2 for 90 days); PU-Chol valve replacement, 4 (30 days); and BOEC-seeded PU-Chol valve replacement, 12 (8 for 30 days and 4 for 90 days).

Explanted BOEC-seeded PU-Chol leaflet implants in general appeared translucent with no gross surface thrombus or other abnormalities on the leaflet (Figs 6A, 6C). By comparison unseeded PU and PU-Chol appeared somewhat opaque and frequently demonstrated grossly visible well-organized white and red thrombi on the surface of the cusps (Fig 6B), which were confirmed to contain activated platelets by immunohistochemistry (Fig 6D). Fluorescent microscopy studies demonstrated that PU-Chol BOEC-seeded cusps in general showed robust retention of BOECs per DAPI fluorescence (Fig 6E); nonseeded explants showed sparse cellularity (Fig 6F). A remarkable flow orientation of the BOEC layer was observed on the seeded explants (Fig 6G). Examination of hematoxylin and eosin stains showed evidence of thrombus on all unseeded explants, but on only one seeded case, and likewise the presence of inflammatory cells on unseeded PU (Fig 6H), which were not observed on seeded explants. No calcifications were noted on any of the microscopic sections.



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  		Fig 6. Appearance of pulmonary polyurethane valve leaflet explants. (A) Gross examination of explants of blood outgrowth endothelial cell–seeded polyurethane-cholesterol leaflets (30 days), showing translucent surfaces with no gross abnormalities, and of (B) 30-day explants of unseeded polyurethane leaflets, showing gross organized thrombi extending to the cusp surface. Immunostaining for CD41/CD61, a marker of activated platelets, showed no immunoreactivity on the intact endothelium of seeded polyurethane-cholesterol 30-day explant (C), but highly positive areas (brown) on unseeded explants (D). (E through G) Fluorescent micrographs of explanted polyurethane leaflets. Nuclei shown by en face 4,6-diamidino-2-phenylindole mount of representative 30-day blood outgrowth endothelial cell–seeded polyurethane-cholesterol (E) and 30-day polyurethane-cholesterol leaflets (F). Phase-contrast bright-field examination of blood outgrowth endothelial cell–seeded explant (G), shown merged with 4,6-diamidino-2-phenylindole fluorescent micrograph of nuclei, showing an organized flow-related orientation of retained blood outgrowth endothelial cells as indicated by white arrow. (H) Hematoxylin and eosin stain of 30-day unseeded polyurethane explant showing only inflammatory cells, including monocyte/macrophages and giant cells. Magnifications of C and D are x650, E–G are x200, and H is x400.

 
Quantitative analyses of the sheep explants demonstrated robust adhesion in both the 30-day and 90-day seeded PU-Chol explants (Fig 7). There was a trend for the cell density to increase comparing 30-day and 90-day results (Fig 7); however, these differences were not statistically significant. However, the unseeded implants showed significantly less cellularity (Fig 7) than did the BOEC-seeded PU-Chol leaflets, and as mentioned above (see Fig 6H), virtually all of the cells noted on the unseeded explant surfaces were inflammatory. Immunostaining of representative BOEC-seeded retrievals demonstrated the uniform presence of von Willebrand factor–positive cells, confirming the persistence of an endothelial phenotype (Fig 8).



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  		Fig 7. Quantitation of cells adhering to pulmonary valve leaflet replacement explants, compared with seeded handling control. Explanted leaflets were 4,6-diamidino-2-phenylindole mounted en face, and the number of nuclei found in representative x200 fields was counted. Blood outgrowth endothelial cell–seeded polyurethane-cholesterol (Chol-PU) leaflets had significantly more cells than unseeded leaflets of any configuration (*p ≤ 0.005), and not significantly fewer than did unimplanted handling controls. (PU = polyurethane.)

 


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  		Fig 8. Characterization of cells adhering to blood outgrowth endothelial cell–seeded polyurethane-cholesterol pulmonary valve leaflet explants. Comparison of cross section of unimplanted blood outgrowth endothelial cell-seeded polyurethane-cholesterol leaflet (A) versus en face 30-day explant (B) and cross section of 90-day explant (C), using immunostaining (brown) for von Willebrand's factor, demonstrating a persistent endothelial phenotype. (Aa), (B), and (Ca) show strongly positive results, compared with negative immunoglobin G controls (Ab and Cb). Magnification x400.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The results of these investigations, both in vitro and in vivo, strongly support the hypothesis that PU-Chol has significantly greater BOEC adhesion properties than unmodified PU. The mechanisms responsible for this may depend on a number of factors, including our earlier observations that PU-Chol was markedly different than unmodified PU with regard to a number of material variables, including increased surface energy, a relatively smoother surface by atomic force microscopy results, and a distinctly different surface chemistry based on Fourier transform infrared spectroscopy comparisons [11]. Taken together all of these material properties are consistent with the view that PU-Chol has a more lipophilic, hydrophobic surface than unmodified PU and thus would have hypothetically a greater affinity for cell membranes. In addition the present studies demonstrated that BOECs cultivated on PU-Chol demonstrate increased collagen synthesis, thereby also implying an increased overall level of extracellular matrix production that should also hypothetically facilitate cell adhesion under high shear. Furthermore, despite the profound differences noted between unmodified and cholesterol-modified polyurethane in vitro and in vivo, the elastomechanical properties of cholesterol-modified polyurethane did not significantly differ from those of unmodified polyurethane (Fig 2). In addition, the presence of cholesterol on the modified polyurethane surface may enable the formation of the integrin–cholesterol–CD47 adhesion complex [19, 20]. It is unknown whether BOECs express CD47, but inasmuch as mature endothelial cells do, it is hypothetically possible that this mechanistic dimension may contribute to BOEC adhesion to PU-Chol under valve level shear forces.

We report the successful BOEC seeding on a PU heart valve leaflet with significantly greater retention of seeded BOECs than controls, both in vitro under simulated valve shear and in vivo. Blood outgrowth endothelial cell seeding of vascular grafts has been investigated experimentally with unfixed heterografts demonstrating successful adhesion in vivo [21]. Thus far there are no reports of BOEC seeding and adhesion in experimental studies with synthetic vascular grafts. Although there have been reports of endothelial seeding of tissue-engineered heart valve leaflet scaffolds [22] using autologous vascular-derived endothelial seeding, thus far there have been no published reports of BOEC seeding of such constructs. This may be because of the relatively less adhesive nature of BOECs compared with mature endothelial cells on synthetic surfaces not modified with cholesterol, as demonstrated in our previous study [11]. Nevertheless, this same study [11] showed that both mature vascular-derived endothelial cells and BOECs demonstrated significantly higher adhesion to PU-Chol versus controls under arterial shear, results consistent with those in the present studies. Thus, PU-Chol, as a result of its highly adhesive properties for BOECs and extracellular matrix mechanisms, represents the most stable seeding substrate investigated thus far for the use of autologous BOECs in synthetic vascular implants.

Previous research from our laboratory investigated postpolymerization derivatizations of polyurethane using bromoalkylation of the urethane nitrogen groups [23, 24]; this same initial reaction step was used in the present studies to prepare PU-Chol. This powerful synthetic approach (ie, bromoalkylation) was successfully used to covalently attach bisphosphonates, which were demonstrated to be efficacious for preventing polyurethane heart valve leaflet calcification in 150-day sheep circulatory studies [24, 25]. The same bromoalkylation approach was used to covalently attach through sulfhydryl linkages anti-adenovirus antibodies for tethering adenoviral gene vectors to the surface of PU heart valve leaflet implants in sheep; successful green fluorescent protein (GFP) reporter transgene expression was demonstrated in white blood cells attaching to the surface of the PU leaflets [18]. Bromoalkylation-mediated cholesterol modification of PU enabled the adhesion of BOECs under heart valve level shear forces both in vitro and in vivo in the present studies. Thus far, multiple derivatizations of PU to impart an array of therapeutic properties have not been attempted. However, it is estimated on the basis of nuclear magnetic resonance data that only 20% or less of available urethane nitrogens have been used in the present cholesterol derivatizations, and thus a considerable reserve of potentially reactive sites remains for potential synergistic combinations of urethane-nitrogen conjugates.

The present studies have several limitations that provide insights for future directions. Calcification was not observed in any of the explants. However, previous investigations from our group have shown that PU leaflet sheep implants of 150 days' duration are required for investigating PU-related pathologic calcification mechanisms [25]. Blood outgrowth endothelial cell seeding of PU-Chol may confer calcification resistance, or if not, the combinatorial use of both PU-Chol and bisphosphonate modifications of PU could be used (as discussed above). In this circumstance, BOEC seeding would still be advantageous for both promoting thromboresistance and preventing inflammatory cell interactions, and hypothetically bisphosphonate derivatization of PU would add calcification resistance. Overall, an implant formulated as such could have major therapeutic advantages. Furthermore, in the present studies, only one seeding density was used, although variations in the density and duration of the seeding protocol may be of interest for future investigations to optimize the time required to prepare a clinical implant. Nevertheless, the period required for the present studies, from cell harvest to seeding and implantation of about 3 weeks, is within a time frame that could work in many clinical scenarios.

In conclusion, PU-Chol has been demonstrated to be a robust substrate for autologous seeding of BOECs for PU heart valve leaflet implants. Polyurethane-cholesterol possesses mechanical properties comparable to unmodified PU, and thus functions comparably in the vascular environment. Furthermore, BOEC adhesion under heart valve level shear forces to PU-Chol is significantly greater than control under simulated and in vivo heart valve shear force conditions. Thus, these results demonstrate successful persistent seeded BOEC adhesion to a prosthetic heart valve leaflet.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
We thank Jennifer LeBold for help in preparing the manuscript. We also thank Ning Dai for technical assistance. This work was supported by the following funding sources: NIH R01-HL59730 and T32-HL07915, and the William J. Rashkind Endowment of the Children's Hospital of Philadelphia.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Farrar DJ, Hill JD. Univentricular and biventricular Thoratec VAD support as a bridge to transplantation Ann Thorac Surg 1993;55:276-282.[Abstract]
  2. Farrar DJ, Lawson JH, Litwak P, Cederwall G. Thoratec VAD system as a bridge to heart transplantation J Heart Transplant 1990;9:415-422.[Medline]
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