doi: 10.1161/CIRCULATIONAHA.105.001446
(Circulation. 2006;114:I-314 – I-318.)
© 2006 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Magnetic Forces Enable Rapid Endothelialization of Synthetic Vascular Grafts
From the Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, Rochester, Minn.
Correspondence to Gurpreet S. Sandhu, Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. E-mail sandhu.gurpreet{at}mayo.edu
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Abstract |
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Background— Synthetic vascular grafts cannot be used in small vessels because of graft failure caused by thrombosis and neointima formation. Rapid endothelialization may overcome this limitation. We hypothesized that a magnetic graft would be able to capture and retain endothelial cells labeled with paramagnetic particles.
Methods and Results— Porcine blood derived endothelial cells were allowed to endocytose superparamagnetic iron oxide microspheres. Cell survival was assessed by trypan blue exclusion and demonstrated a dose-dependent cell survival of 75% to 95%. A flexible magnetic sheet was annealed to the external surface of a knitted Dacron graft. Labeled cells (106/mL) were placed within the graft for 5 minutes. Confocal and electron microscopy confirmed uniform cell capture at the magnetized surface. The effect of shear forces on the adherent cells was evaluated in a flow chamber. The cells remained attached at rates up to 300 mL/min, with cell loss commencing at 400 mL/min. Prototype magnetic grafts were implanted in porcine carotid arteries. Labeled cells were placed within the graft for 10 minutes at the time of implantation. The grafts were evaluated after one day and uniform cell coverage was noted on the magnetized surface. In comparison, relatively few labeled cells were seen attached to a nonmagnetized surface.
Conclusions— Magnetic forces can be used to rapidly cover a vascular graft with paramagnetically labeled cells. This biophysical interaction is sufficient to retain cells in the presence of blood flow. Applications of this technique may include rapid endothelialization of synthetic vascular grafts and dialysis fistulas.
Key Words: coronary disease • endothelium • grafting • surgery • magnet
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Introduction |
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The major limitation of prosthetic vascular grafts is their tendency to occlude after various periods of time. This occlusion rate is higher for smaller-diameter grafts and precludes their use in a significant number of medical applications, most notably in coronary artery bypass grafting. Numerous studies have shown that failure is secondary to graft occlusion, either because of thrombogenicity of the synthetic material or because of encroachment of tissue (intimal hyperplasia) into the lumen of the graft at anastomotic sites.1 A potential way to limit graft failure would be to provide rapid, uniform, and complete coverage with a functional endothelial layer. In a pioneering study, Stump et al have shown that a Dacron patch suspended in the flow, without contact with the vessel wall, was covered with endothelial colonies within 7 days of implantation.2 Early efforts at graft endothelialization with the use of mature endothelial cells,3–8 although promising, were limited by difficulties related to obtaining cells in significant numbers. The recent description of circulating endothelial progenitor cells9 has provided a new source for cellular seeding of grafts. We have previously shown that blood-derived endothelial outgrowth cells (EOCs) are effective in preventing restenosis and can restore vascular function in animal models of arterial injury.10 Previous work used prolonged vascular occlusion to enable cell adhesion to the vessel wall, an approach that cannot be used in clinical settings. We hypothesized that local cell capture and retention could be accomplished by using magnetic forces. EOCs were rendered magnetically attractable by loading them with superparamagnetic microspheres and a prototype magnetic graft was developed. Cell capture by magnetic interaction is likely to facilitate rapid and uniform cellular coverage of vascular grafts and alleviate some of their present shortcomings.
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Methods |
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Generation and Characterization of EOCs
Peripheral blood mononuclear cells were isolated from 50 to 200 mL porcine blood by Ficoll density gradient (Histopaque 1077; Amersham) as previously described.10–12 Briefly, cells were placed on fibronectin-coated plates and grown in EBM-2 culture media supplemented with EGM-2 SingleQuots (Cambrex Bio Science). The presence of a highly proliferative population with endothelial characteristics (EOCs) was noted after 7 to 10 days. EOCs were plated on fibronectin-coated slides for analysis of endothelial features. For detection of von Willebrand factor (vWF), cells were fixed with methanol, permeabilized with 0.1% Triton X-100—phosphate-buffered saline (PBS) solution and blocked with 10% normal serum. Primary sheep anti-human vWF (The Binding Site) followed by donkey anti-sheep biotin/streptavidin-FITC were used. Isotype identical IgG (Sigma) served as control. Lectin binding was tested with Griffonia Simplicifolia Lectin I isolectin B4 (Vector Laboratories).
Cell Labeling
Superparamagnetic microspheres (SPMs) (0.9-mm diameter, 63.4% iron oxide, precoated with a green fluorescent tag; Bangs Labs) were added to EGM-2 media at various concentrations for the in vitro studies. For in vivo experiments, SPMs were added to media containing cultured EOCs at a 500:1 SPM-to-cell ratio. Cells were incubated at 37°C for 16 hours, then labeled with a second fluorescent tag by incubation for 30 minutes at 37°C with the red carbocyanine tag CM-DiI (Molecular Probes). Cells were then washed 3 times with PBS, trypsinized, and resuspended in PBS at 106 cells/mL for subsequent use.
Effects of Iron-Loading on Cell Survival
EOCs were incubated with escalating doses of SPM particles (range, 0 to 4000 SPM-to-cell ratio). Cell viability was evaluated by trypan blue exclusion performed after 16 hours of incubation. The number of apoptotic cells was evaluated with the TUNEL method (In Situ Cell Death Detection Kit, TMR red; Roche) performed at 4, 24, and 48 hours after exposure. Apoptotic cells were counted on 10 random high-power fields and the results averaged. All experiments were performed at least in triplicate.
Development of a Magnetic Dacron Graft
Commercially available knitted Dacron grafts (Boston Scientific) of 6- to 8-mm diameter were used. Rectangular pieces were cut from commercially available magnetic rubber sheets. Their surface magnetic strength was &100 gauss as measured by a gauss meter. The rectangular pieces were glued to the external surface of the Dacron graft, providing a tubular structure that had &50% of its circumference covered by the magnetic sheet. The grafts were gas-sterilized for in vivo experiments.
Cell Capture
Grafts were cut longitudinally into magnetic and nonmagnetic parts. The graft pieces (magnetic and nonmagnetic) were placed at the bottom of wells of 6-well culture dishes, EOCs (106/graft) were added, and the plates were placed on an orbital shaker at 30 rpm speed. After 5 minutes of exposure, the grafts were removed, carefully washed with PBS, and examined en face with a confocal or fluorescent microscope. Presence of CM-DiI–positive cells was considered conclusive of EOC capture; SPM green fluorescence was obscured by a large amount of autofluorescence from Dacron. Cell capture was quantified by averaging the number of CM-DiI–positive cells on 10 high-power fields selected at random on the graft surface. All experiments were performed at least in triplicate.
Effect of Pulsatile Flow on Cell Capture
Grafts were filled with EOC suspension (106/graft), clamped for 5 minutes, then connected to a flow system driven by a peristaltic pump. PBS was added to the system, and circulated at flows of 100 mL/min, 200 mL/min, 300 mL/min, and 400 mL/min. Fluid dynamic viscosity 
was 0.0014 Pa·sec (1.4 centiPoise; Sonoclot, Sineco, Inc), yielding Reynolds numbers between 146 and 583 (Re =2Rv
/; R=radius, v=mean velocity, =density). This would suggest presence of laminar flow, and it allowed estimation of shear rate and wall shear stress based on Hagen-Poiseuille equation at 79 to 314 sec–1, and 0.11 to 0.44 N/m2 (1.1 to 4.4 dyne/cm2), respectively. Because blood flow was pulsatile, it can be inferred that peak wall shear stress was actually higher than our calculations. These values are within the range of wall shear stress measured in large arteries.13 After 30 minutes of flow, the grafts were removed, cut into magnetic and nonmagnetic parts, and inspected en face under a fluorescent microscope. Cell capture was quantified by averaging the number of CM-DiI–positive cells on 10 high-power fields selected at random on the graft surface. All experiments were performed in triplicate for each flow rate.
In Vivo Experiments
Domestic pigs weighing 40 to 80 kg were used in these studies. A total of 200 mL blood was obtained under sedation 2 to 3 weeks before intervention and used for generation of autologous EOCs. Cells were incubated overnight with SPM particles at a 500:1 microsphere-to-cell ratio and labeled with CM-DiI on the day of the experiment. Animals were sedated with a combination of ketamine and xylazine, and anesthetized with isoflurane. The skin was prepped and draped in sterile fashion. The graft placement was performed under the supervision of an experienced vascular surgeon (M.K.). The common carotid arteries were dissected free and the animals received heparin bolus (60 U/kg intravenous) and additional doses to maintain activated clotting time greater than 200 seconds. The common carotid was clamped proximally and distally, and a straight segment without side branches was resected. The grafts (8x30 mm, N=2; 6x30 mm, N=6) were filled with EOC suspension (106/graft) for 10 minutes before placement. The magnetic strip extended 20 mm along the graft length to allow room for suturing, and it covered &50% of the circumference. This allowed presence of both a magnetic surface and a nonmagnetic control surface within a single vessel. The graft was sutured end-to-end with 6.0 Prolene wire, carefully de-aired, and circulation restored. Presence of blood flow distal to the graft was verified with a Doppler probe. The dissection planes were sutured in layers and the animals were allowed to recover. All animals received aspirin on the day of the procedure and on the postoperative day and subcutaneous heparin 8 hours after intervention. After 24 hours the grafts were removed, inspected for presence of thrombus, washed in PBS, and longitudinally cut into magnetic and nonmagnetic halves. Cell retention was quantified by averaging the number of CM-DiI–positive cells on 10 high-power fields selected at random on the graft surface. All animal protocols were performed in accordance with the position of the American Heart Association on laboratory animal use and were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic.
Statistical Analysis
Statistical analysis was performed using the SAS software. The normal distribution was tested with the Shapiro-Wilk statistic, and transformations were performed when appropriate. Results were analyzed with 1- or 2-way ANOVA, as appropriate; Tukey t test was used for multiple pairwise comparisons. In the case of non-normal distributions, the Kruskal-Wallis test (overall effect) and the Wilcoxon rank-sum test (pairwise comparisons) were used. P<0.05 was considered significant. All calculations were performed with the SAS procedures GLM, UNIVARIATE, NPAR1WAY. Data are presented as mean±SD.
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
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Results |
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Results of the effects of SPM particle loading on EOCs are given in Figure 1. There was a significant increase in trypan blue inclusion with increasing SPM to EOC ratio (Figure 1). All SPM doses except 500:1 ratio induced a statistically significant increase in the number of trypan blue positive cells (P<0.01 versus nontreated cells). SPM loading at 500:1 ratio was not different from nontreated cells (5.6±0.6% versus 7.7±2.9%; P=not significant). Although a dose-related response is suggested in the Figure, differences between higher doses (1000:1, 2000:1, and 4000:1) were not statistically significant. The apoptotic effects of SPM loading were significantly higher at 24 hours than at 4 and 48 hours for all doses tested, suggesting that apoptosis is a transient event. Based on these results, and on preliminary experiments showing that a 500:1 ratio provides sufficient EOC loading to allow magnetic attraction and capture, this dose was selected for subsequent experiments. The ability to bind lectin and expression of vWf were consistent with an endothelial phenotype of EOCs used in this experiment (Figure 2).
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The magnetic forces generated through the Dacron graft were sufficient to attract cells to the inner surface. Presence of a magnetic surface increased cell capture 30-fold (264±20.5 versus 8.9±3.0 cells/high-power field; P<0.01). When placed in pulsatile flow, EOC retention was >70% up to flow rates of 300 mL/min, with a statistically significant decline to 61% in attached cells occurring at 400 mL/min (Figure 1); conditions were comparable with flow in large arteries in vivo. Representative images from confocal microscopy, as well as an image from flow chamber experiments, are shown in Figure 2.
A total of 8 grafts were placed in carotid interposition in normal swine. In the first 2 implants, there was a sizable mismatch between the 8-mm Dacron grafts and carotid artery diameter (estimated at 4 to 5 mm), which required careful and gradual tapering of graft ends before placement. At 24 hours, 1 of these 8-mm grafts was subtotally occluded with thrombus. The remaining 7 grafts (1 with 8-mm and 6 with 6-mm diameter) were all widely patent, without any thrombus. En face microscopy showed that labeled cells were present in substantial amounts on the magnetized surface, but in small numbers on the nonmagnetized control (435±153 versus 38±31 cells/high-power field; P<0.001). Representative images from an explanted prototype graft are shown in Figure 2.
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Discussion |
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This is the first article to our knowledge to report the use of biophysical forces to attach endothelial cells to the surface of prosthetic vascular grafts. EOCs readily endocytose paramagnetic particles and are rapidly attracted to magnetized graft surfaces. Once seeded, these cells remain attached at shear forces comparable to those seen in the circulation. A porcine study provided proof of concept and demonstrated the presence of a large number of seeded cells after 24 hours of pulsatile blood flow.
Vascular grafts based on either expanded polytetrafluoroethylene or polyethylene terephthalate fiber (Dacron) work very well when positioned in vessels with diameters >6 mm and in conditions of high flow. However, the majority of vascular disease occurs in vessels with caliber of <6 mm. Coronary bypass grafting is commonly performed with arterial conduits (when available), and with autologous saphenous vein grafts. However, up to 30% of the patients do not have veins suitable for vascular reconstruction because of abnormalities and inadequate quality, or may lack vessels because of previous surgery.14 Low-flow situations found in venous reconstruction or arterio-venous fistulas further predispose these grafts to occlusion because of intimal hyperplasia at anastomotic sites.15 Therefore, the development of reliable small caliber prosthetic grafts would greatly expand surgical options in these situations.
Pioneering work on endothelialization was performed as early as the late 1970s, when Herring et al first reported on autologous endothelial cell seeding.3 Endothelial cell seeding was shown to be effective in reducing graft thrombogenicity and intimal hyperplasia in several animal models.4–8 Promising results in human trials were reported initially by Ortenwall.16 In these studies, one limb of an aortic Dacron bifurcation prosthesis was seeded with autologous endothelial cells harvested from the saphenous vein, whereas the other limb was sham-seeded with culture medium only. However, these results were not reproducible by other groups.17–19 The disadvantage of the 1-stage seeding technique using veins as a source is that only a limited number of cells can be extracted, ranging from 0.56x105 to 100x105.20 Using the 2-stage "sodding" technique has improved clinical results, but requires long-term endothelial cell culture (4 to 6 weeks).21,22
A second obstacle in endothelialization of grafts is the poor retention of seeded cells. Human endothelial cells show little adhesion to the currently available vascular graft materials. Rosenman et al have shown 70% cell loss from the surface of a PTFE graft within first 30 minutes of exposure to pulsatile flow; with a slower exponential loss occurring over the next 24 hours.23 To overcome this limitation, chemical coatings (collagen, fibronectin, laminin, poly-l-lysin, gelatin, etc), pre-clotting of the graft (blood, plasma, serum, fibrin glue), and surface modification (heparin, arginine-glycine-aspartic acid, lectins, cell adhesion peptides) have been proposed.24 The limitation is that most coatings will induce activation of the coagulation mechanisms, thereby limiting potential beneficial effects. A different approach is to coat with materials that will allow in vivo capture of endothelial progenitor cells from the circulation. However, initial reports with a CD34 antibody covered graft were disappointing.25
In our experiments, we chose a completely different approach. First, we used EOCs cultured from peripheral blood as opposed to invasive tissue harvesting described to obtain endothelial cells from veins. EOCs are a well-characterized, uniform population of cells and proliferate rapidly10,11 within 2 to 3 weeks of culture. An alternative to these cells would be to use culture-modified mononuclear cells, which can be grown in significant numbers in as few as seven days and have similar vasculoprotective effects.
Second, we have used a novel biophysical approach for cell capture and retention. This is a significant departure from previous seeding/sodding/capturing techniques in which the surface of the vascular graft had to be covered with various materials. In our approach, the magnetic material is applied on the outside surface of the graft, thereby leaving the luminal surface unaltered. We were able to show that EOCs are easily loaded with SPMs, and that they are rapidly captured and retained on to the Dacron graft surface under pulsatile flow conditions. Our animal experiments confirmed that the labeled EOCs were present in significant numbers 24 hours after grafting. Interestingly, EOCs were present in larger numbers on the nonmagnetized graft side in vivo than under in vitro flow conditions, possibly because of better adherence to Dacron in the presence of blood components. However, cell retention was on average 30-fold higher on the magnetized surface (range, 4- to 100-fold on randomly selected high power fields). Paramagnetic particle loading had little effect on cell viability at the dose used for in vivo experiments.
In these initial studies, we were able to demonstrate a proof-of-principle for magnetic capture of cells to arterial grafts. The present study was limited to 24 hours because magnetic materials with a biocompatible profile suitable for long-term experiments are not readily available. Long-term experiments will be a requisite step for further development. An obvious goal for these studies is application in small diameter grafts, an active area of research focus.
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Acknowledgments |
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We extend appreciation to Traci Paulson for secretarial support and Laurel Kleppe for technical expertise.
Source of Funding
This work was supported in part by National Institutes of Health grant HL75566 (R.D.S.).
Disclosures
None.
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Footnotes |
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Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.
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