in vivo performance of dual ligand augmented endothelialized expanded polytetrafluoroethylene...

In Vivo Performance of Dual Ligand Augmented EndothelializedExpanded Polytetrafluoroethylene Vascular Grafts

Bernard P. Chan,1 Wenge Liu,1 Bruce Klitzman,2 William M. Reichert,1 George A. Truskey1

1Department of Biomedical Engineering, Duke University, Hudson Hall, Room 136, Box 90281,Durham, North Carolina 27708-0281

2Kenan Plastic Surgery Research Laboratories, Duke University, Durham, North Carolina 27708-0281

Received 4 February 2004; revised 3 May 2004; accepted 10 May 2004Published online 6 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30114

Abstract: In this study, we examined combinations of three approaches to improve theadhesion of endothelial cells (EC) onto expanded polytetrafluoroethylene (ePTFE) vasculargrafts placed at the femoral artery of rats: (1) high-affinity receptor-ligand binding ofRGD–streptavidin (SA) and biotin to supplement integrin-mediated EC adhesion; (2) cellsodding to pressurize the seeded EC into the interstices of the ePTFE grafts; and (3) longerpostseeding attachment time from 1 to 24 h prior to implantation. An in vitro system, whichaccounts for cell loss due to both graft handling and shear stress, was designed to optimizeconditions for in vivo experiments. Results suggest that longer in vitro attachment time enabledthe adherent EC to endure mechanical stresses by forming strong adhesions to the underlyingextracellular matrix substrates; cell sodding helped to retain the adherent EC by physicallydocking the cells against the graft interstices; and the SA–biotin interaction enhanced theearly attachment of EC but did not lead to better cell retention or reduced surface coverageof blood clot in the current study. Mechanical manipulation of cells during implantation is alimiting factor in maintaining a confluent EC layer on synthetic vascular grafts. © 2004 WileyPeriodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 72B: 52–63, 2005

Keywords: blood–material interaction; cell–material interactions; endothelialization; sur-face modification vascular graft

INTRODUCTION

In cardiac and peripheral bypass surgery, atherosclerotic re-gions of vessels are replaced by autologous veins or arter-ies.1,2 Nevertheless, some patients do not have appropriatevessel replacement due to prior removal, disease, or inade-quate size. This has prompted the need for vascular grafts asreplacements. In larger blood vessels, with diameter greaterthan 6 mm, synthetic vascular grafts have worked reasonablywell.3 Clinical application of small diameter, less than 6 mm,synthetic prostheses continue to meet with difficulties, suchas thrombus formation in the short term and intimal hyper-plasia in the long term.4 Given the anticoagulant properties ofendothelial cells (EC), these problems may be overcomepartially if grafts are seeded with an adherent monolayer ofautologous, functional EC prior to implantation. Practicalsuccess of this method has been limited by autologous ECharvesting and viability, graft handling, and poor cell reten-tion after exposure to flow conditions.5–7

Efforts to overcome these difficulties have involved twoprimary approaches. First is a tissue-based approach thatattempts to recreate vessel constructs based on coculturing ofEC and smooth muscle cells.8,9 However, tissue engineeringof multilayered cell-based vessels is still in its infancy, and itspracticality in clinical applications is limited by time to growthe grafts and expense.10 Second is a biocompatibility ap-proach that aims to improve the blood compatibility of thegraft material by enhancing the efficiency of luminal ECseeding. Improvement of EC attachment has been accom-plished through coating the graft surface with cell-adhesiveproteins such as fibronectin (Fn), albumin, gelatin, collagen,fibrin glue, laminin, REDV peptide, and RGD peptide.11–17

These approaches, however, have limitations, in that protein–integrin bonds have relatively low affinity (�106 M�1).18 Theseeded cells therefore do not develop mature attachments ina short period of seeding time, and cell loss was still signif-icant following restoration of shear flow when Fn was pread-sorbed onto the synthetic graft surface.19

To ensure rapid and strong initial attachment, we devel-oped a “dual ligand” system in which streptavidin(SA)–biotin, a high affinity (Ka � 1010–1013 M�1) ligand-receptorpair, supplemented the lower affinity Fn–integrin (Ka � 106–

Correspondence to: G.A. Truskey (e-mail: [email protected])Contract grant sponsor: NIH; contract grant number: HL-44972

© 2004 Wiley Periodicals, Inc.

52

109 M�1) binding of EC to synthetic surfaces.20–24 The rapidformation of the SA–biotin linkages stabilizes the EC andpromotes the initial Fn–integrin bond formation. Once theseinitial bonds form, the cell initiates cytoskeletal changes thatcause the cell to spread, increasing the surface area exposedto the substrate. This process brings the cell membrane re-ceptors in the vicinity of the ligands on the substrate, thusfacilitating integrin mediated bond formation. With the dualligand system, a higher percentage of cell retention is main-tained, and the area of focal contact increases significantlycompared to control Fn single-ligand system.20–22 The use ofthe dual-ligand system also accelerated the adherent EC toreach the quiescent state that may have additional advantagesin reducing graft thrombogenecity.23 In the presence of phys-iological shear stress, the production of nitric oxide andprostacyclin were significantly enhanced, and the adhesion ofleukocytes onto EC surfaces was inhibited.23

EC sodding is another approach that accelerates the for-mation of an EC monolayer on synthetic grafts.25 Intralumi-nal pressure is applied to force the seeded EC into theinterstices of prosthetic grafts. Microvascular EC soddingresulted in significantly more cells adherent to the luminalsurface of grafts at the time of implant.25–29 Satisfactoryresults were demonstrated in both 4-mm diameter25–28 and1-mm diameter vascular grafts.29

The current study examined whether adhesion of humanumbilical vein EC onto 1-mm internal diameter (ID) ePTFEcould be improved by combinations of dual ligand, cellsodding, and prolonged static attachment. To assess in vivoperformance, the 1-mm ID ePTFE graft was implanted asshort interpositional femoral graft in a rat. Both cell soddingand SA–biotin EC coupling strategies are employed in theexperiments, and postseeding attachment times of 1 and 24 hwere examined. The implanted grafts were examined forpatency 15 min after restoration of blood flow. The graftswere then explanted, fixed, and examined for EC retentionand blood clot surface coverage.

The in vivo studies employed rats that are a standardmodel for ePTFE graft thrombosis assessment.30,31 The fem-oral artery graft was selected as the implantation site becauseof its superficial location and long length with few branch-es.32 The study was designed to examine the acute responseof the EC-seeded ePTFE grafts by quantifying the initial cellloss after 15 min of blood flow. These short-term optimiza-tion studies will serve as the basis for future long-termstudies.

MATERIALS AND METHODS

ePTFE Graft and Cell Treatment

One-milliliter ID ePTFE vascular grafts with a 30 �m meaninternodal distance (Impra Inc., Tempe, AZ) were steamsterilized and denucleated of air by immersing in 95% ethanolfor 15 min, and then washed with distilled water. Denucle-ation increases the patency of small diameter grafts in

vivo33,34 by forcing the ePTFE fibrils to be in contact with anaqueous solution,33 thus increasing the surface area availablefor cell binding and protein adsorption.

Human umbilical vein endothelial cells (HUVEC, Clonet-ics Inc., Walkersville, MD) were grown to confluence ingelatin-coated T75 flasks with Endothelial Basal Medium(EBM, Clonetics Inc.) supplemented with EndothelialGrowth Medium (EGM), growth factors (Clonetics Inc.), andfetal bovine serum (Sigma). Cells in passage 2 to 4 were usedfor all experiments. Flasks of confluent HUVEC were washedthree times with phosphate-buffered saline (PBS, pH 7.2) anddetached using trypsin and diluted with EBM to give a celldensity of 2 � 106 cells/mL. For the Fn control system,suspended cells in nonserum EBM without further modifica-tion were seeded onto the ePTFE graft pretreated with Fnusing a rotating device as described below. For the SA–biotinsystem, suspended cells in nonserum EBM were incubatedwith 50 �g/mL of RGD–SA in PBS for 40 min and subse-quently seeded onto the ePTFE graft pretreated with a mix-ture of Fn and biotinylated albumin b-BSA. RGD–SA is a giftfrom Dr. Patrick Stayton from the University of Washington.Expression and purification of RGD–SA are described else-where.22,35

Protein Coating and Graft Endothelialization

Two approaches were used to achieve graft endothelializa-tion: EC seeding and EC sodding. Uniform EC seeding andprotein coating on the ePTFE graft was accomplished byrotating the graft at 1 rpm (Fig. 1). The 1-mm ID ePTFE graftwas placed along the center axis, with one end connected toa 1-mL syringe via a 22-gauge needle and the other endsecurely clamped. To optimize the graft surface for HUVECadhesion, surfactant and cell adhesion proteins were adsorbedon the graft surface before the graft endothelialization. First,2.5 mg/mL tridodecylmethyl ammonium chloride (TDMAC)was injected for 1 h as the first layer of coating. The cationic

Figure 1. Schematics of the rotating apparatus designed to achieveuniform cell seeding density. The rotation speed was set at 1 rpm. The1-mm ID ePTFE graft, with EC cell suspension injected within itslumen, was placed along the center axis with one end connected toa 1-mL syringe via a 22-gauge needle and the other end closed witha microvascular clamp.

53STREPTAVIDIN–BIOTIN AUGMENTED ENDOTHELIALIZED ePTFE

surfactant TDMAC has been found to enhance protein ad-sorption on hydrophobic surfaces such as ePTFE.33,34 Next, amixture of 15 �g/mL Fn with or without 2 mg/mL b-BSAwas injected for 1 h. Last, HUVEC suspended in nonserumEBM at an initial concentration of 2 � 106 cells/mL with orwithout RGD–SA was injected for 1 h. Rotation was startedimmediately after each injection for the entire 1-h duration.The rotational adhesion procedures were performed inside anincubator with an atmosphere of 5% CO2 and 95% air at37°C. The surface density of Fn and BSA attached to theePTFE lumen was measured with a radiolabeling method aspreviously described.24 The surface Fn density, biotin recep-tor density, and RGD–SA density were determined by theHABA assay (Sigma) and an 125I iodination labeling method(Pierce, IL) as previously described.20

The cell-sodding apparatus is illustrated schematically inFigure 2. The ePTFE graft was securely clamped at one end,while the other end was connected to a 60-mL syringe via a22-gauge needle. The syringe was attached to a syringepump. EBM was flushed through the graft interstices and intoa container underneath. The intraluminal pressure, as mea-sured with the manometer, was set at 5 psi throughout thesodding procedure. To evaluate the effect of cell sodding onthe relative location of the adherent cells in the ePTFE graft,both nonsodded endothelialized grafts and cell-sodded graftswere fixed in 10% formalin in PBS, dehydrated in gradedethanols, paraffin embeded, cross-sectioned, and stained withhematoxylin and eosin. Photomicrographs were obtained on aNikon Optiphot light microscope.

In Vitro Examinations

To optimize conditions for in vivo experiments, implantationgroups were selected based on results obtained using an in

vitro system designed to simulate the physical manipulationencountered during surgery and the shear stress effect by theflowing blood. A total of eight treatment groups were exam-ined.

1. Treatment 1: Fn 1-h attachment: EC seeded on the Fntreated ePTFE graft and attached for 1 h.

2. Treatment 2: dual-ligand 1-h attachment; RGD–SA incu-bated EC seeded on the dual-ligand–treated ePTFE graftand attached for 1 h.

3. Treatment 3: Fn 24-h attachment; EC seeded on the Fn-treated ePTFE graft and attached for 24 h. After the 1-hattachment, the cell-adherent graft was placed in a 35-mmtissue culture dish and into the incubator for another 23 h.Leaving the cell-seeded graft on the rotating device formore than a few hours would deplete all the nutrients fromthe media as the graft lumen could only hold small amountof media. For 1 h, both the media and cells were in goodcondition as judged by the cell morphology and the colorof the media that indicates optimal pH (data not shown).

4. Treatment 4: dual-ligand 24-h attachment; RGD–SA-in-cubated EC seeded on the dual-ligand–treated ePTFE graftand attached for 24 h.

5. Treatment 5: Fn 1-h attachment plus sodding; adherent ECsodded onto the Fn-treated ePTFE graft for 5 min follow-ing the 1-h attachment.

6. Treatment 6: dual-ligand 1 h attachment plus sodding;RGD–SA-incubated EC sodded onto the dual-ligand–treated ePTFE graft for 5 min following the 1-h attach-ment.

7. Treatment 7: Fn 24 h attachment plus sodding; adherentEC sodded onto the Fn-treated ePTFE graft for 5 min after

Figure 2. Schematics of the sodding apparatus. ePTFE graft was securely clamped at the open end,while the other end was connected to a 60-mL syringe via a 22-gauge needle. The syringe wasattached to a syringe pump. EBM media was flushed through the graft interstices and into a containerunderneath for 5 min. The intraluminal pressure, as measured with the manometer, was set at 5 psithroughout the sodding procedure.

54 CHAN ET AL.

the 1-h attachment, followed by an additional 23 h of staticincubation.

8. Treatment 8: dual-ligand 24-h attachment plus sodding;RGD–SA incubated EC sodded onto the dual-ligand–treated ePTFE graft for 5 min after the 1-h attachment,followed by an additional 23 h of static incubation.

To simulate the surgical conditions and identify limitingsteps, vascular clamps were placed at the two ends of the 7mm-long ePTFE graft after each of the eight treatments.Every 10 min, the position of the clamp was slightly adjustedto simulate the suturing movements. Fresh EBM media, with2% v/v HEPES buffer added, was used to cover the entiregraft, and the media was replenished every 5 min as liquidcontinuously dripped off during surgery and had to be replen-ished every few minutes. The above procedures were re-peated for 1 h, the approximate time for anastomosing theePTFE graft during surgery, and they were performed outsidethe sterile hood under a heat lamp to simulate the heating padused and the body heat of the rat during surgery.

Upon the completion of the surgery simulation experi-ments, the flow system shown in Figure 3 was used to mimicthe shear stress created by flowing blood. The flow systemconsisted of a 2-cm length of the 1-mm ID ePTFE graft, adual syringe pump (Harvard Apparatus, Holliston, MA), 5%CO2 percolated media reservoir, and a pulse dampener (ColeParmer, Vernon Hills, IL) was inserted between the syringepump and the cell-seeded graft to minimize shear stress

fluctuations. Shear stress, �, was calculated from Poiseuille’sequation:

� �4�Q

�r3 (1)

where Q is the volumetric flow rate (cm3/s), � is the mediaviscosity, and r is the radius of the cylindrical graft. Theapplied � was estimated to be 15.3 dynes/cm2 using literaturevalues for the viscosity of rat blood (0.04 poise36) and theflow rate in the rat femoral artery (2.25 mL/min37). All flowexperiments were performed at 37°C for 15 min, and eachexperiment was performed three times.

At the conclusion of in vitro endothelialization treatments1–8, simulated surgery, or in vitro flow experiments, graftswere fixed with 10% formalin in PBS, cut open, and treatedwith 4,6-diamidino-2-phenylindole (DAPI, Sigma) nuclearstain for cell counting. The DAPI-stained images provideadditional information on cell distribution on the experimen-tal graft surface. For the cell count procedure, six randomlocations from the DAPI-stained piece were inspected forendothelial cell coverage. Fluorescent Images (as shown inFig. 6) were obtained using an inverted microscope equippedwith epifluorescent filter with excitation wavelength of 365nm and emission wavelength of 425 nm. The images werethen processed and analyzed using NIH image software. Cellcount was obtained by manually summing the nuclei in eachof the six random fields. Cell density was computed bydividing the nuclei count number with the total image area.

Figure 3. Schematics of the in vitro flow system. The flow system consisted of a 2-cm length of the1-mm ID ePTFE graft, a dual syringe pump, 5% CO2 percolated media reservoir, and a pulsedampener to minimize pulsation and fluctuations in shear stress.


Prostacyclin Measurement

To examine the effect of different treatments on the func-tional state of EC, prostacyclin produced by EC under in vitrotreatments 1–8 was measured using the 6-keto-prostaglandinF1� enzyme immunoassay (EIA) system (Amersham LifeScience, IL). Upon the completion of each treatment, thecell-seeded graft was placed in a six-well plate covered with2 mL of EBM media. A volume of 50 �L media was thentaken for the EIA measurement, followed by trypsinizing thecells on the ePTFE graft for cell counting using a hemocy-tometer. Each experiment was performed three times.

Graft Implantation

Animal care and use complied with the PHS Guide for theCare and Use of Laboratory Animals, USDA regulations (9CFR Parts 1,2,3), and the Federal Animal Welfare Act (7USC 2131 et. Seq.). The protocol was approved by the DukeUniversity Institutional Animal Care and Use Committee(Registry Approval # A333-97-7).

Male CD rats (weight range 280 to 450 g; Charles RiverLabs, Raleigh, NC) were anesthetized with sodium pentobar-bital (35 mg/kg, i.p.). The ventral side of the rat was shavedand the femoral arteries were exposed. Blood flow in theartery was occluded by placing two microsurgical clamps onthe artery, separated by approximately 7 mm. The artery wasthen transected and a 7-mm length of treated ePTFE graft wasanastomosed as an interpositional graft using standard micro-surgical technique with nylon 10-0 monofilament suture(Ethicon, New Brunswick, NJ) and an operating microscope.After suturing both the proximal and distal anastomoses, theclamps were removed and blood flow was reestablished in thesutured graft for a duration of 15 min of graft implantationtime. Throughout the surgery, fresh EBM media, with 2% v/vHEPES buffer added, was applied to the exposed graft inorder to stabilize the pH.

For the in vivo experiments, six groups were examined: (1)bare ePTFE grafts with no adhesion proteins and no adherentcells; (2) ePTFE grafts adsorbed with b-BSA and Fn butwithout adherent cells; (3) EC seeded and attached for 1 h onFn-treated ePTFE; (4) EC seeded and attached for 1 h on dualligand-treated ePTFE; (5) Fn-treated ePTFE graft with ECsodding and 24 h attachment; (6) dual- ligand–treated ePTFEgraft with EC sodding and 24-h attachment. Groups 1 and 2were used as controls to examine the effect of the adsorbedcell adhesive protein on graft thrombosis. Groups 3 and 4were selected to examine the effectiveness of endothelializedvascular grafts under the clinically relevant 1-h period ofgraft treatment.21,25 Groups 5 and 6 were selected based onresults obtained from the in vitro experiments.

Patency Test

The patency of the implanted grafts was confirmed by twostandard tests.37 First, the distal artery was gently lifted untilthere was a partial occlusion (“flicker test”). With each cycleof the heart, the artery would partially open and reocclude,

indicating the graft was patent. Second, the artery proximal tothe graft was clamped and the distal section was gentlymilked with forceps to push the remaining blood distally(“milking test”). The proximal clamp was then removed andthe distal artery is observed. If it filled up with blood, the graftwas considered patent. Each graft was required to pass bothtests at the time of explantation to be considered patent.

Quantification of EC Attachment and Retention

After 15 min of in vivo flow, the grafts were explanted, gentlywashed with Ringer’s solution for 5 min, fixed with 10%formalin in PBS for 10 min, and cut along the long axis toproduce two pieces for analysis of the luminal surface. Onepiece was subjected to DAPI nucleus staining for cell count-ing as explained in previous section, and the other piece wassubjected to SEM.

Scanning Electron Microscopy

Grafts were fixed with 10% formalin in PBS, rinsed with afew drops of EBM, and dehydrated by serial dilutions ofethanol (30, 50, 70, 80, 90, and 100%) for 10 min each, thencritical point drying was achieved by adding a few drops oftrimethylsilane (TMS) to each sample until the TMS evapo-rated. The samples were mounted on aluminum stubs usingdouble-stick tape, and sputtered with gold-paladium using avacuum evaporation apparatus. Specimens were examined ina Phillips SEM 515 using a tilt angle of 60 degrees andelectron energies of 20 kV. Cell growth of ePTFE wasassessed qualitatively by comparing the cell spreading andcell density.

Quantification of Surface Clot Coverage

The luminal surface of explanted grafts were photographedwith a digital camera. The total graft area and the blood clotcovered area of the luminal graft surface were determinedusing NIH image after normalizing illumination using thebackground intensity.

Statistical Analysis

Instat 2.0 was used to statistically compare data to accesssignificant variations. Student’s t-test or one-way ANOVAwith Tukey post test was conducted to determine p values.All data were reported as means � SD.

RESULTS

Protein and Cell Density on ePTFE Graft

Surface densities of the protein adsorbed to the ePTFE graftlumen were comparable to the values measured when pro-teins were adherent on glass.22 As summarized in Table I,treatments of ePTFE with TDMAC and denucleation signif-icantly promoted the adhesion of Fn and b-BSA onto the graftsurface by two to three orders of magnitude. RGD–SA den-

56 CHAN ET AL.

sity on cells was measured at 3.1 � 0.1 � 109 molecules/cm2.Rotational EC seeding and attachment produced an evenlydistributed layer of adherent cells on the ePTFE graft. Rep-resentative images are shown in Figure 6. The rotationalprocess also enhanced the efficiency of EC seeding. Initialcell density resulted from the various in vitro treatments weresummarized in Table II. At 1-h attachment, EC density was6.3 � 0.9 � 102 cells/mm2 for the Fn control, a sixfoldimprovement over our previous attempt in which we seededthe ePTFE graft by manually rotating the sample.21 The useof SA–biotin significantly increased the 1-h EC density to8.3 � 1.1 � 102 cells/mm2. Attachment for 24 h againincreased EC initial density to 9.3 � 1.4 � 102 cells/mm2 and11.5 � 1.6 � 102 cells/mm2 for Fn and dual-ligand cases,respectively. In contrast, cell sodding did not lead to higherinitial cell density in all examined cases.

Effect of Pressure Sodding

Throughout the sodding procedure, fluid penetrated the inter-stices of the graft and was observed as fluid droplets beading up

on the outer graft surface. The procedure did not result in anyobserved bleeding (in vivo experiments) or fluid leakage (in vitroexperiment) through the graft interstices. All incidents of bleed-ing were limited to the anastomotic sites at the time blood flowwas reestablished, and the bleeding stopped instantaneously.Fluid penetration during sodding forced the adherent cells intothe graft interstices and caused cells to penetrate to a depth ofapproximately 50 �m into the graft wall (Fig. 4).

Results from In Vitro Experiments

Previous experience indicates that cell detachment from theendothelialized synthetic vascular graft is caused by a com-bination of fluid shear stress and the handling of the graftsduring microsurgery.21 The in vitro system described in thecurrent study characterized both phenomena independently.Table II summarizes EC retention under the eight in vitrotreatment conditions, perturbed first by handling and then by15.3 dynes/cm2 of fluid shear stress for 15 min. Cell densityand retention on the dual ligand treated surfaces were ingeneral higher than the Fn controls, but were significantly

TABLE I. Effect of TDMAC and Graft Denucleation on BSA, Biotin, and Fn Densities on the ePTFE Graft

BSA Densitymolecules/cm2

Biotin Densitymolecules/cm2

Fn Density,molecules/cm2

With TDMAC and Denucleation (Fn) — — 1.9 � 0.1 � 1011*†

With TDMAC and Denucleation (Dual Ligand) 7.9 � 0.6 � 1010* 1.5 � 0.1 � 1011* 1.6 � 0.1 � 1011*Without TDMAC and Denucleation (Fn) — — 1.5 � 0.1 � 108†

Without TDMAC and Denucleation (Dual Ligand) 4.0 � 0.3 � 108 7.2 � 0.5 � 108 9.6 � 0.8 � 107

The surface Fn and BSA densities attached on the ePTFE were measured with a radiolabeling method. HABA assays were used to quantify the surface biotin receptor as wellas the RGD-SA density. Reported values are averages of three separate measurements.

* Denotes a significant enhancement effect ( p � 0.001) due to TDMAC and graft denucleation.† Indicates the difference in Fn density between the Fn control surface and the dual ligand surface is significant ( p � 0.05).RGD-SA density on Cell Membrane � 3.1 � 0.1 � 109 molecules/cm2.Biotin Density on Glass � 2.1 � 0.3 � 1012 molecules/cm2.20

Fn Density on Glass � 1.6 � 0.1 � 1011 molecules/cm2.20

TABLE II. Initial Cell Density, Cell Retention, and Prostacyclin Production of the Cell-Seeded ePTFE Graft under Different In VitroConditions

Treatment

Initial CellDensity,

102 Cells/mm2Cell Retention

after Handling, %

Cell Retentionafter Handlingand Flow, %

Prostacyclinproduction underStatic Condition,

�M/106 cells

1: Fn 1-h attachment 6.3 � 0.9 46.0 � 10.9 32.8 � 11.4 0.02 � 0.0022: Dual-ligand 1-h attachment 8.3 � 1.0# 49.1 � 13.9 38.0 � 10.7 0.02 � 0.0023: Fn 24-h attachment 9.3 � 1.4* 65.7 � 11.2 60.4 � 9.6* 0.06 � 0.006*4: Dual-ligand 24-h attachment 11.5 � 1.6* 72.1 � 13.5 64.7 � 10.0* 0.06 � 0.004*5: Fn 1-h attachment � sodding 7.1 � 0.9 59.8 � 12.4 46.6 � 8.4 0.03 � 0.0046: Dual-ligand 1-h attachment � sodding 8.6 � 0.8 62.1 � 11.8 45.5 � 9.2 0.03 � 0.0047: Fn 24-h attachment � sodding 11.3 � 1.5* 83.7 � 15.4* 75.2 � 11.2*† 0.05 � 0.004*†

8: Dual-ligand 24-h attachment � sodding 11.9 � 1.6* 81.9 � 15.0* 76.5 � 8.9*† 0.05 � 0.005*†

Initial cell density was obtained by counting adherent cells on the ePTFE graft immediately after the respective treatment (N � 3). Cell retention was determined by comparingthe cell density before and after the surgical perturbation and the application of shear stress. Perturbations that simulated surgical ligation of the ePTFE graft were performed asdescribed in the Methods section. Shear stress was applied using the setup shown in Figure 3. Prostacyclin release was determined by measuring the level of 6-keto-prostaglandinin the media normalized by cell number. Reported values are averages of three experiments (N � 3).

# indicates that at 1-h attachment, the dual ligand had significantly higher initial cell density than the Fn control ( p � 0.05).* Indicates the measured value was significantly higher than the attachment control ( p � 0.05).† Denotes that the measured value was significantly higher than the 1-h attachment plus sodding case ( p � 0.05).


different in only one instance, the initial cell density at 1-hattachment.

Regarding the effects of graft handling (Table II, column3), the addition of cell sodding had only a modest andinsignificant increase in cell retention when EC attached for1 h prior to handling. Increasing cell attachment time to 24 hwithout cell sodding also produced insignificant increases incell retention. However, increasing cell attachment to 24 htogether with cell sodding, significantly improved cell reten-tion to better than 80%.

The effect of flow following graft handling (Table II,column 4) followed a similar trend on EC retention; however,significant increases in EC retention were observed for 24-hattachment without sodding and for 24-h attachment withsodding, both cases yielding approximately 50% increase incell retention. Clearly, 24-h attachment plus sodding yieldedthe best results independent of the use of the dual-ligand orthe Fn control surfaces, retaining greater than 70% of cells.

Cells adherent for 1 h on either Fn or dual-ligand producedidentical amounts of prostacyclin, 0.02 � 0.002 �M/106

cells. Cells adherent for 24 h on either Fn or dual ligandproduced significantly higher levels of prostacyclin, 0.06 �0.007 �M/106 cells (p � 0.05). At 24-h attachment, ECseeded on ePTFE produced significantly more prostacyclinthan EC seeded on glass slide (p � 0.05), which was mea-sured previously at 0.03 � 0.002 �M/106 cells.23 Cell sod-ding did not have significant effect on the prostacyclin pro-duction at either attachment time point (Table II, column 5).

Results from In Vivo Experiments

Six different groups, as described in the Materials and Meth-ods section, were examined for in vivo patency. After 15 minof blood flow, patency tests revealed that Group 1 had apatency rate of 75% (three out of four), Group 2 was 50%(two out of four), and Groups 3–6 had patency rates of 100%(20 out of 20). Figures 5 and 6 contain SEM and DAPI-stained images comparing EC coverage for Groups 3–6 takenbefore and after 15 min of blood flow. As shown in the twofigures, panels (E) and (F), cell detachment was substantialafter 15 min of in vivo flow for the 1-h attachment cases. Incontrast, panels (G) and (H) of Figures 5 and 6 show that themajority of cells remained adherent after 15 min of in vivoflow for the 24-h attachment and sodding cases. Under higherSEM magnification, more red cells, platelets, and leukocyteswere observed on graft area that was depleted of adherent EC[Fig. 5(I)].

Cell densities before and after the reestablishment of invivo blood flow were quantified with the DAPI-stained mi-crographs. Blood clot coverage was quantified by graft “red-ness” using images of the explanted grafts shown on Figure7. Table III summarizes the cell retention and the blood clotcoverage for all examined cases. As expected, 24-h attach-ment and sodding resulted in significantly higher cell reten-tion as compared to the 1-h attachment cases. Consistent withthe in vitro results, differences between the dual-ligand casesand Fn control cases were not significant. Clot coverage wassignificantly more prominent for the nonendothelialized cases[Fig. 7(A–B)]. Clot coverage data in Table III confirmed thisobservation.

DISCUSSION

The current study was designed to examine the initial cell lossfrom EC-seeded ePTFE grafts during surgery and the acuteonset of fluid flow for 15 min. The study employed an in vitrosystem that (1) mimics the in vivo implantation procedure,and (2) applies similar magnitude of shear stress across thecell-seeded ePTFE graft as for flowing blood. We identifiedthat graft handling is more significant than flow in causing ECdetachment, and EC retention is improved by using a cellsodding method and a longer time frame for postseedingattachment. After 15 min of in vitro flow or in vivo implan-tation, grafts treated with sodding and 24-h attachment ex-hibited an average of 75.9 � 14.3% EC retention in vitro and66.2 � 11.5% in vivo. These values were significantly betterthan those of the 1-h attachment. The in vivo blood clotcoverage of 16.9 � 10.9% was also significantly lower thanthe 65.5 � 6.9% of the nonendothelialized group. The dem-onstrated improvements in cell retention and antithrombo-genic potential provide preliminary evidences for the feasi-bility and effectiveness of the EC-seeded small diametersynthetic vascular graft.

The graft ligation procedure appeared to disturb the ad-herent EC layer on the graft lumen. Cell loss from graft

Figure 4. Effect of sodding on the adherent EC. (A) Hematoxylin andeosin-stained section of the nonsodded control graft. Cells were adher-ent on the ePTFE surface for 24 h. (B) Hematoxylin and eosin-stainedsection of the sodded graft. EC were pressurized into the interstices withthe sodding apparatus and incubated for 24 h. Bar � 25 �m.

58 CHAN ET AL.

handling was especially significant at the short postseedingattachment time of 1 h in which more than half of theadherent cells were detached during implantation (Table II).The extent of cell loss was more significant due to grafthandling (52.5 � 17.7%) than due to shear stress alone

(25.6 � 23.6%). This limitation prompted the use of cellsodding and a longer postseeding cell attachment time.

EC sodding has been reported as a method of acceleratingthe formation of an antithrombogenic cell lining on the lu-minal surface of prosthetic vascular grafts,25–29 as well as

Figure 5. Scanning electron photomicrograph of the adherent cells before (A–D) and after (E–H) the15 min of in vivo implantation. (A) and (E) are representative images of EC adherent on the dual ligandsurface for 1 h. (B) and (F) represent EC adherent on the Fn surface for 1 h. (C) and (G) are images ofEC sodded on the dual ligand surface and adherent for 24 h. (D) and (H) are images of EC sodded onthe Fn surface and adherent for 24 h. (I) is a magnified image of the bare graft area showing thepresence of red cells, leukocytes, and platelets.


dramatically reducing the time necessary to deposit EC on theluminal surface of the synthetic grafts.25,27,28 Sodding alsohelps to retain the adherent EC by pressuring them into thegraft interstices.25–29 As shown in Figure 4, cells were forcedinto the luminal surface of the graft to a depth of approxi-mately 50 �m. Consequently, cell loss due to graft handlingfrom sodded grafts was reduced to 39.0 � 17.1% for the 1-hattachment cases, and to 17.2 � 21.5% for the 24-h attach-ment cases (Table II).

Allowing the adherent EC to incubate and attach on thegraft surface for longer period of time enabled the adherentEC to develop mature focal adhesion contacts and actin stressfibers.24 As a result, the adherent cells could better withstandthe mechanical perturbation endured during graft handling, asdemonstrated by the improvement in cell retention noted in

Table II. Another potential advantage of using a longer ad-hesion time is the antiplatelet activity conferred by the ad-herent EC. At 24 h of attachment, adherent EC becomeantithrombotic and release nitric oxide and prostacyclin whenshear stress was encountered.23 The release of these agentsmay then reduce platelet aggregation and fibrin formation.We found that at 24 h of cell attachment, EC release signif-icantly more prostacyclin than the 1 h cell attachment cases.The enhanced prostacyclin production did reduce blood clotcoverage on the ePTFE graft from an average of 26.5 �11.3% (1-h attachment cases) to 16.9 � 10.9% (24-h attach-ment cases), but the difference was not statistically signifi-cant.

Aside from the cell adhesion treatments, we also enhancedthe cell retention indirectly by modifying the graft condition

Figure 6. DAPI-stained pictures of the ePTFE graft before (A–D) and after (E–H) the 15 min of in vivoimplantation. (A) and (E) are representative images of EC adherent on the dual ligand surface for 1 h.(B) and (F) represent EC adherent on the Fn surface for 1 h. (C) and (G) are images of EC sodded onthe dual ligand surface and adherent for 24 h. (D) and (H) are images of EC sodded on the Fn surfaceand adherent for 24 h.

60 CHAN ET AL.

and surface chemistry. To facilitate protein binding, we de-nucleated the ePTFE graft and treated the graft lumen withTDMAC. Data from the Fn and BSA density quantificationsupported the use of these graft treatments (Table I). Further-more, we achieved uniform cell seeding density with a rota-tional procedure (Figs. 5 and 6). A uniform layer of EC may,in turn, improve the graft patency. Seeding EC by simplyturning the graft at an angle for a certain period of timeencourages the formation of cell clusters in one area but baregraft in others, subsequently leading to graft failure.21

One unexpected result was that the SA–biotin and Fndual-ligand system did not further enhance adhesion. Wehave previously demonstrated in vitro on plain glass surfacesthat the use of dual-ligand significantly enhanced EC attach-ment both at the short-term 1-h and the long-term 24-hattachment period.20–24 In the current study, the effect ofSA–biotin was limited to the 1-h attachment cases in whichthe dual ligand significantly promoted the initial EC densityover the Fn control. Differences between the dual-ligand andFn control were insignificant in all other comparisons (TablesII and III). There are several likely reasons for the results.

First, mechanical manipulation to cells from handling thegraft during surgery may weaken the strength of cell adhesionto an extent that the advantage gained from using the dualligand was rendered insignificant. Second, the use ofTDMAC may not produce as firm adhesion as b-BSA. Por-tions of cells may thus detach via the TDMAC-ePTFE linkthat is independent of the EC-substrate interaction. Third,relative to the plain glass surface, the placement and orien-tation of the b-BSA substrate may not be as uniform on thecylindrical ePTFE graft. Proteins may be embedded into thegraft interstices and positioned away from the adherent EC.Finally, the effect of the dual ligand may be overridden bysodding and/or prolonged attachment time. Sodding en-hanced EC retention by physically docking the cells againstthe graft interstices, whereas prolonged attachment time al-lowed the cells to form more bonds to have stronger adhe-sion. These enhancements in cell adhesion may dominate theevents of higher affinity binding.

In conclusion, we demonstrated in this study various meth-ods from graft treatments to cell adhesion strategies thatresulted in significant improvement in cell retention as well as

Figure 7. Photographs showing the ePTFE grafts under different conditions immediately after beingexplanted. Graft in (A) was treated with protein substrates b-BSA and Fn only with no adherent EC.Graft in (B) was without protein substrates and EC. (C) shows the graft in which EC were adherent onthe Fn surface for 1 h. (D) shows the graft in which EC were adherent on the dual ligand surface for1 h. (E) shows the graft in which EC were sodded and adherent on the Fn surface for 24 h. (F) showsthe graft in which EC were sodded and adherent on the dual ligand surface for 24 h.


reduction of blood clot coverage on the ePTFE surface. Weidentified that graft handling is a significant event that causescell detachment. The problem can be ameliorated by thecombined use of cell sodding and longer postseeding cellattachment time. Some of the graft treatments in this studywere not as promising as we had anticipated, especially intheir effect on cell retention and clot coverage. Longer in vivoimplantation period or higher shear stress regimen may pro-vide some improvements. The cell retention results in thisstudy were a significant improvement over our previous at-tempt,21 although there was still more than 30% cell loss inour best case.

We thank Todd McDevitt and Patrick Stayton of the Universityof Washington for providing the RGD-SA mutant, and Kevin Ol-brich of Duke University for advice and discussions.

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TABLE III. Luminal Coverage of the ePTFE Graft underDifferent In Vivo Conditions

Treatment Group

CellRetention,

%

ClotCoverage,

%

Group 1:1-h Attachment (Fn, N � 4) 26.4 � 6.0 29.4 � 7.4

Group 2:1-h attachment (dual ligand,

N � 4) 33.8 � 7.3 23.6 � 8.5Group 3:

24-h Attachment � sodding(Fn, N � 6) 65.1 � 8.1* 18.4 � 7.5Group 4:

24-h Attachment � sodding(dual ligand, N � 6) 67.2 � 8.2* 15.3 � 7.9Group 5:

Bare surface, N � 4 — 65.5 � 6.9†

Group 6:Protein-only surface (b-BSA

� Fn), N � 4 — 95.8 � 6.2†‡

Groups 1–4 are conditions with adherent EC. Cell retention is determined bycomparing the cell density on the explant to the cell density right after the respective cellseeding treatment. Clot coverage is determined by quantifying the area of blood clot onthe graft explant (Fig. 7) with NIH image.

* Indicates that the 24-h attachment and sodding cases had significantly better ( p �0.05) cell retention than the 1-h attachment cases.

† Denotes that surfaces without EC had significantly higher ( p � 0.05) clotcoverage than the EC-seeded cases.

‡ Indicates the effect of protein coating on clot coverage of the ePTFE graft issignificant ( p � 0.05).

62 CHAN ET AL.

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