Journal Article Critique one-page typed single spaced (11 or 12 point font)

The blood and vascular cell compatibility of heparin-modiﬁed ePTFE vascular grafts Ryan A. Hoshi a, Robert Van Lith a, Michele C. Jen a, Josephine B. Allen b, Karen A. Lapidos a, Guillermo Ameer a,c,* aBiomedical Engineering Department, Northwestern University, Evanston, IL 60208, USAbMaterial Science and Engineering Department, University of Florida, Gainesville, FL 32611, USAcDepartment of Surgery, Feinberg School of Medicine, Chicago, IL 60611, USA article info Article history:

Received 16 July 2012 Accepted 21 September 2012 Available online 12 October 2012 Keywords:

Vascular graft Elastomer Endothelial cell Progenitor cell Smooth muscle cell Heparin Hemocompatibility Aminated poly(1,8-octanediol-co -citrate) (POC) abstract Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and therefore have higher rates of complications that involve thrombosis and restenosis. We developed an approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to ami- nated poly(1,8-octanediol- co-citrate) (POC) via its carboxyl functional groups onto POC-modi ﬁed ePTFE grafts. The bioactivity and stability of the POC-immobilized heparin (POC eHeparin) were characterized via platelet adhesion and clotting assays. The effects of POC eHeparin on the adhesion, viability and phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were also investigated. POC eHeparin grafts maintained bioactivity under physiologically relevant conditions in vitro for at least one month. Speci ﬁcally, POC eHeparin-coated ePTFE grafts signi ﬁcantly reduced platelet adhesion and inhibited whole blood clotting kinetics. POC eHeparin supported EC and BOEC adhesion, viability, proliferation, NO production, and expression of endothelial cell-speci ﬁc markers von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured on POC eHeparin showed increased expression of a-actin and decreased cell proliferation. This approach can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity and improved endothelialization are desirable properties. 2012 Elsevier Ltd. All rights reserved. 1. Introduction Cardiovascular disease is a leading cause of death and morbidity in developed countries and patients diagnosed with this disease often require revascularization via bypass grafts to restore blood ﬂ ow to tissue. Autologous vein bypass grafts, the gold-standard of care, cannot always be harvested, prompting the use of prosthetic vascular grafts. However, prosthetic vascular grafts have poor long- term patency, particularly when used in small diameter applica- tions [1,2]. Therefore, there remains an urgent need for safe and effective materials for the fabrication of vascular grafts. Heparin and other anticoagulants have been incorporated into biomaterials to inhibit intrinsic thrombogenicity [3e7] . Heparin immobilization strategies have included physisorption, electro- static deposition, and covalent bonding to surfaces [3,4,6,8,9]. Forexample, cross-linked collagen surfaces have been used for the covalent immobilization of heparin to improve blood compatibility, but the use of highly thrombogenic materials such as collagen for vascular grafts may be problematic [4,10]. Other strategies include the controlled release of heparin from electrospun materials but soluble heparin can lead to increased risk of heparin-induced thrombocytopenia, which can be fatal in some circumstances [11] . To further augment vascular graft thromboresistance, some promising approaches have combined heparin immobilization with other known anti-clotting agents such as nitric oxide (NO) or thrombomodulin, but these multi-factorial approaches are complex and may be cost prohibitive for commercialization [6,9].

Our lab has reported the synthesis of citric acid-based biomate- rials for use in tissue engineering applications [12e19 ] . In particular, poly(1,8-octanediol- co-citrate) (POC) is an elastomeric polyester that can be used to form composites, fabricated to have micro and nano-architectures, and used as a coating to modify medical devices to potentially improve their performance [14,16e21] . In previous studies by our group and others, POC was demonstrated to be * Corresponding author.

E-mail address: [email protected] (G. Ameer). Contents lists available atSciVerse ScienceDirect Biomaterials journal homepage: www.elsevier .com/locate/biomaterials 0142-9612/$esee front matter 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.biomaterials.2012.09.046 Biomaterials 34 (2013) 30 e41 a biocompatible material with good hemocompatibility and minimal acute and chronic inﬂammatory responses in vivo [14,16,19,22] . POC also has been shown to support endothelialization under physiological ﬂow conditions in vitroandin vivo [13,15].

Furthermore, the available carboxyl and hydroxyl groups on POC provide options to chemically modify the polymer network with macromolecules to engineer new functionality. The quest to develop a functional vascular graft would bene ﬁt from a simple, effective and safe method to incorporate the antithrombogenic and anticoagulant properties of heparin into a biomaterial. In this regard, the effects of immobilized heparin on vascular cell processes should also be understood. Herein, we describe the functionalization of POC with heparin and investigate the effects of POC-immobilized heparin on whole blood clotting, platelet adhesion, and the adhesion, viability, and phenotype of endothelial cells and smooth muscle cells. As circulating progenitor cells can contribute to the endothelialization and arterial healing process, the effect of POC eHeparin on blood outgrowth endothelial cells (BOECs) is also investigated. BOECs are the progeny of bone marrow-derived circulating endothelial progenitor cells and emerging evidence supports their use in cell-based therapies for promoting postnatal vasculogenesis and clinical use in cardiovas- cular applications [23,24]. Differentiation of these cells may promote the formation of a healthy endothelium on vascular graft surfaces [13,25,26] . This work also investigates whether immobi- lized heparin will have a negative effect on smooth muscle cell proliferation.

2. Materials & methods 2.1. Synthesis and preparation of POC All reagents and chemicals were purchased from Sigma eAldrich (St. Louis, MO) unless noted otherwise. The synthesis of POC has been previously described [19].

Brie ﬂy, equimolar amounts of citric acid and 1,8-octanediol monomers were melted under a ﬂow of nitrogen gas at 160 e16 5 C and then the temperature of the system was lowered to 140 C to create a pre-polymer. Subsequently, the pre-polymer was puri ﬁed in water, freeze-dried and then reconstituted in ethanol. The pre-polymer was post-polymerized in tissue culture polystyrene (TCP) multiwell plates at 80 C for 4 days. To fabricate POC-coated vascular grafts, thin-walled ePTFE tubes (6 mm inner diameter, 30 e50 mm internodal distance, Zeus Inc., Orangeburg, SC) were coated with 10% pre-polymer using a previously described spin-shearing method and post-polymerized at 80 C for 4 days [13]. The POC coating on the ePTFE was veri ﬁed visually using SEM and toluidine blue dye, since it can bind with high af ﬁnity to negatively charged carboxylic acid functional groups [27].

2.2. Heparin immobilization to POC POC samples were rinsed extensively in PBS at 37 C for several days to remove unreacted monomers which may interfere with the conjugation chemistry. Prior to conjugation, POC-coated plates/vascular grafts were soaked in 0.1 MMES buffer (pH 5.6, containing 0.5 MNaCl) for 1 h. POC was covalently modi ﬁed using standard carbodiimide chemistry. To covalently bind the carboxyl groups of POC to the dia- minohexane intermediate, a solution of 50 mM DH solution was prepared with 300 mM N-(3 dimethylaminopropyl)- N 0-ethylcarbodiimide (EDC) and 150 mM N -hydroxysuccinimide (NHS) catalysts in 0.1 M2-(N -morpholino) ethanesulfonic acid (MES) buffer, pH 5.6, containing 0.5 MNaCl and incubated with POC for 5 h at room temperature. The POC was extensively rinsed with 2 MNaCl and water to remove excess reagents. The immobilized DH was conjugated to the carboxylic acid groups of heparin by incubating the POC with heparin solution (2.5e 5 mM) (heparin sodium salt isolated from porcine intestinal mucosa, MW 15 kDa, 167 units/mg) with 100 mM NHS and 200 mM EDC catalysts in MES buffer overnight at room temper- ature. A concentration of 5 mM heparin solution was determined to be the maximal solution concentration during conjugation as determined previously by toluidine blue assay (data not shown) and used for blood compatibility and cell compatibility studies [28]. Excess reagents and non-covalently bound heparin were removed by extensive washing with 2 MNaCl solution and water. For cell compatibility studies, samples were sterilized by exposure to ethylene oxide gas.

2.3. Immobilized heparin quantiﬁ cation and contact angle measurements The presence of heparin bonded to POC-coated ePTFE grafts was detected and quanti ﬁed using the metachromatic dye, toluidine blue, as described previously [28] . The surface density of the POC eHeparin ePTFE coating was evaluated for stability after 14 days and 28 days of incubation in vitroat 37 C in PBS and compared against freshly prepared samples. To control for background dye binding, the POC e Heparin samples were compared against incubation-matched controls consisting of POC conjugated with diaminohexane. Static water-in-air contact angle measure- ments were taken over time using a custom-built contact angle goniometer (components from Rame-Hart, Inc., Mountain Lakes, NJ). A total of three different samples were measured for each surface type: ePTFE, POC eePTFE and POC eHeparin ePTFE over a 15-min period.

2.4. Heparin detection by X-ray photoelectron spectroscopy (XPS) Elemental analysis using X-ray photoelectron spectroscopy (XPS) was per- formed to verify the incorporation of diaminohexane and heparin by conﬁ rming the presence of nitrogen and sulfur peaks. POC eHeparin samples were prepared using 2.5 mM heparin solution during conjugation. XPS spectra were collected using Omicron ESCALAB (Omicron, Taunusstein, Germany) with a monochromated Al K a (1486.6 ev) 300 W X-ray source. Measurements consisted of broad survey scans as well as high-resolution N(1s) and S(1s) scans.

2.5. Isolation of blood outgrowth endothelial cells from human peripheral blood The isolation and characterization of BOECs obtained from peripheral blood have been previously described by our lab [13]. Brie ﬂy, forty- ﬁve milliliters of peripheral blood were collected from adult volunteers in the presence of acid citrate dextrose (ACD, Solution A; BD Biosciences). All procedures involving blood collec- tion were performed in accordance with the regulations of the Northwestern University Institutional Review Board. Peripheral blood mononuclear cells (PB-MNCs) were isolated from whole blood via histopaque density gradient centrifugation using Accuspin tubes (Sigma Aldrich, Milwaukee, WI). The isolated PB-MNCs representing the starting populations were suspended in endothelial cell (EC) growth medium-2 (Lonza, Baltimore, MD) without the fetal bovine serum supplement but with 5% allogeneic human serum (Lonza). The PB-MNCs were seeded onto ﬁbronectin-coated plates (BD Biosciences; San Jose, CA), and cultured at 37 C in a humidi ﬁed incubator containing 5% CO 2. After 4 days, non-adherent cells were removed by complete media change, and media was changed every 3 e4 days thereafter. BOECs were detected as tightly packed colonies with the characteristic cobblestone morphology of endothelial cells. After colony isolation, BOECs were expanded onto TCP and maintained through eight passages.

2.6. Bioactivity and antithrombogenicity assessment of POC eHeparin ePTFE grafts 2.6.1. Re-calci ﬁed whole blood clotting The anti-clotting properties of POC eHeparin surfaces were assessed using modi ﬁed re-calci ﬁed plasma and whole blood clotting assays [13,15]. Whole blood was collected from adult volunteers into ACD anticoagulant (BD Biosciences, Franklin Lakes, NJ). Sections of ePTFE ePOC eHeparin grafts (5 mm in length) were pre-weighed and placed in 1.5 mL centrifuge tubes. As a control, non-modi ﬁed ePTFE and POC-coated ePTFE samples were used. The anticoagulated whole blood samples were re-calci ﬁed with the addition of 10% (v/v) 0.1 MCaCl 2and then 750 mL of re-calci ﬁed blood were then immediately incubated with graft samples for 1 h at room temperature. The grafts and all clotted blood were carefully removed, brie ﬂy blotted on a paper towel and weighed. The bioactivity of POC eHeparin ePTFE coating was evaluated after 14 days and 28 days of incubation in vitroat 37 C in PBS.

To evaluate the heparin bioactivity after adsorption with plasma proteins, samples were incubated with platelet poor plasma for 1 h at 37 C prior to the whole blood clotting assay. Previous plasma protein studies have determined that a majority of protein becomes adsorbed to heparin-coated surfaces within this time frame [29].

To account for the variability between blood donor collections, each blood clotting experiment included a set of non-modi ﬁed ePTFE control samples to normalize the whole blood clot mass for POC and POC eHeparin experimental samples.

2.6.2. Platelet adhesion Whole blood was collected from adult volunteers into ACD anticoagulant, which has previously been reported to preserve platelet activity [13]. The blood was centrifuged at 250 g for 15 min to obtain platelet-rich plasma (PRP) supernatant. The PRP preparation and platelet suspension buffer (PSB) used for this experiment were described previously [30]. Samples of ePTFE were cut into disks using a cork borer to match the surface area of 96 multiwell plates and gently pinned down to remain in place. PRP, diluted 1:10 in PSB, was incubated at 37 C for 60 min with prepared ePTFE samples and gently rinsed with warm PBS. The number of adherent platelets was determined by detecting the amount of lactate dehydrogenase (LDH) present after cell lysis as previously described [15]. Brie ﬂy, adherent platelets were lysed by incubation with 2% Triton-PSB buffer for 30 min at 37 C. A colorimetric substrate for LDH (Roche Diagnostics Corporation, Indianapolis, IN) was added and incubated for 20 min at 37 C. The reaction was stopped with the addition of 1 Nhydrochloric acid.

The optical density was measured at 490 nm with a reference wavelength of 650 nm.

A calibration curve was generated from a series of serial dilutions of a known platelet concentration and used to determine the number of adhered platelets. The R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 31 morphology of adhered platelets was assessed via scanning electron microscopy (SEM). Brieﬂy, adherent platelets were ﬁxed using 2.5% glutaraldehyde in PBS for at least 2 h, dehydrated in a graded series of ethanol, and freeze-dried. The samples were then sputter-coated with a 7-nm layer of gold and observed using scanning electron microscopy (SEM 3400N, Electron Probe Instrumentation Center, North- western University).

2.7. Effect of immobilized heparin on vascular cells Human umbilical vein endothelial cells (HUVEC) (Lonza, Baltimore, MD) (passages 3 e6) were cultured in EGM-2 media. Human aortic smooth muscle cells (HASMC) (Lonza) (passages 3 e5) were cultured in SmGM-2 media. BOECs were isolated and cultured as described earlier. All cells were cultured at 37 Cin a humidi ﬁed incubator containing 5% CO 2. HUVECs and HASMCs (seeding density 1 10 4cells/cm 2) were seeded onto POC, POC eHeparin and TCP surfaces. Similarly, the seeding density for BOECs was 7.5 10 3cells/cm 2. Cell culture media was changed every 3 days.

2.7.1. Cell proliferation At predetermined time intervals, cells were lysed and quanti ﬁed using a Pico- Green DNA assay (Molecular Probes, Carlsbad, CA) and compared against standards of known cell numbers.

2.7.2. Cell viability Cell viability of adherent cells was assessed using a live/dead cell viability assay kit (Invitrogen, Carlsbad, CA) after 4 days of culture. Following manufacturer ’s instructions, after incubation with the live/dead staining solution, the cells were gently rinsed in warm PBS and adherent cells were imaged for viability by ﬂuo- rescence microscopy.

2.7.3. Cell phenotype Cells were ﬁxed with 4% paraformaldehyde and blocked with 10% normal goat serum. HUVECs and BOECs were probed with primary antibodies to EC-speci ﬁc markers von Willebrand factor (vWF) (Dako Cytomation, Carpenteria, CA) and CD144 (VE-Cadherin) (R&D Systems, Minneapolis, MN). HASMCs were probed with a-actin smooth muscle (abCam, Cambridge, MA). Cells were counterstained with Hoechst and analyzed by ﬂuorescence microscopy (Nikon TE2000U). POC and POC eHeparin surfaces have background ﬂuorescence when stained with Hoechst; therefore, the background ﬂuorescence was digitally removed for clarity.

2.7.4. Nitric oxide production HUVECs and BOECs were cultured on TCP, POC and POC eHeparin surfaces for up to 4 days and then probed for production of nitric oxide using the ﬂuorescent probe 5,6-diamino ﬂuorescein diacetate (DAF-2 DA, Santa Cruz Biotechnology, Santa Cruz, CA) [31]. After cell detachment with trypsin-EDTA, cell suspensions were incubated with 5 mM DAF-2 DA for 1 h at 37 C. The ﬂuorescence of DAF was excited at 488 nm and emitted ﬂuorescence was measured at 530/40 nm using a BD LSR2 ﬂ ow cytometer (BD Biosciences, San Jose, CA).

2.8. Statistical analysis Numerical data are reported as mean standard deviation (SD). The statistical signi ﬁcance between two sets of data was calculated using a two-tail Student ’s t -test. One way and two way ANOVA tests were used to measure differences for experiments with multiple data sets with a post hocBonferroni test performed between groups with signiﬁ cant differences to correct for the multiple pairwise comparisons. A value of p< 0.05 was considered to be statistically signiﬁ cant. 3. Results 3.1. Heparin immobilization to POC The covalent modi ﬁcation of thin POC ﬁlms with diaminohex- ane and heparin was assessed by XPS for N1s and S1s spectra ( Fig. 1 ). The presence of nitrogen from diaminohexane amine functional groups was detected for POC ediaminohexane in addi- tion to POC eHeparin surfaces. As expected, no nitrogen or sulfur peaks were detected for unmodi ﬁed POC surfaces and no sulfur Fig. 1. Detection of immobilized heparin by X-ray photoelectron spectroscopy. XPS spectra for N(1s) and S(1s) for POC, POC conjugated with DH (POC eDH), and POC eHeparin. Fig. 2. Schematic of the bioactive POC eHeparin ePTFE vascular graft.

R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 32 peaks were detected for POCeDH surfaces. Surfaces conjugated with heparin showed peaks for sulfur.

3.2. Fabrication of the POC eHeparin ePTFE graft Vascular grafts were prepared by coating ePTFE tubes with a coating of POC using a “spin shear ”coating technique developed in our lab [13]. The POC coating was used to prepare the ePTFE for subsequent covalent immobilization of heparin via a DH linker molecule ( Fig. 2). The POC pre-polymer is cross-linked within the node and ﬁbril structure of the ePTFE lumen at 80 C. The POC and POC eHeparin coatings are capable of modifying the ePTFE without signi ﬁcantly altering the original node and ﬁbril architecture ( Fig. 3).

The presence of heparin on the luminal surface of modi ﬁed ePTFE grafts was con ﬁrmed visually by a purple color change from the toluidine blue stain ( Fig. 3D). The cationic toluidine blue dye can also bind to negatively charged carboxyl groups present in POC and showed signi ﬁcant background staining for POC-coated ePTFE. The purple color change was only seen on POC eHeparin ePTFE. No dye binding was observed for ePTFE control grafts. The toluidine blue dye was also used to quantify the surface density of POC eHeparin- coated ePTFE grafts and con ﬁrm the stability of the immobilized heparin after incubation under physiological conditions in vitro ( Fig. 3 E). There were no statistically signi ﬁcant changes in the heparin surface density over time ( p¼ 0.58). The heparin surface density after incubation for 14 days and 28 days was 35.5 11.1 and 37.4 8.9 ng/mm 2, respectively, compared with 47.2 14.6 ng/ mm 2for freshly prepared samples.

Static water-in-air contact angle measurements also con ﬁrmed the successful modi ﬁcation of the ePTFE luminal surface with a coating of POC and surface immobilized heparin. After a 5-min time period, the POC eHeparin ePTFE had a signi ﬁcantly reduced water-in-air contact angle compared with ePTFE and POC eePTFE surfaces ( Fig. 3F). After 15 min, ePTFE, POC eePTFE and POC e Heparin ePTFE had water contact angles of 98.43 5.32 , 86.67 6.53 and 20 18 .17 , respectively. Fig. 3. Characterization of POC eHeparin vascular grafts. SEM micrograph of the luminal surface of (A) unmodi ﬁed ePTFE, (B) POC-coated ePTFE and (C) POC eHeparin-coated ePTFE.

Arrows indicate areas with POC-coated ﬁbrils (scale bars: 20 mm). (D) POC eHeparin coating on ePTFE grafts was determined by toluidine blue staining showing a purple color change, en facepreparations of graft segments show lumen side up. (E) Heparin surface density on freshly prepared (Day 0) POC eHeparin-coated ePTFE grafts and after 14 days and 28 days incubation in vitroat 37 C in PBS, N.S. ¼“not signi ﬁcant ”,n¼ 6, mean SD. (F) Static water-in-air contact angle measurements for ePTFE, POC eePTFE and POC eHeparin- coated ePTFE grafts. * p< 0.05, signi ﬁcantly less than ePTFE, ** p< 0.01, signi ﬁcantly less than ePTFE and POC, *** p< 0.001 signi ﬁcantly less than ePTFE and POC, n¼ 4, mean SD.

[For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.] R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 33 3.3. Bioactivity and antithrombogenicity assessment of POCe Heparin ePTFE grafts 3.3.1. Re-calci ﬁed whole blood clotting The bioactivity of POC eHeparin ePTFE grafts was assessed over a 28-day incubation in vitroat 37 C in PBS. A re-calciﬁ ed whole blood clotting assay demonstrated the potent anticoagulant activity of the POC eHeparin ePTFE grafts ( Fig. 4). The POC eHeparin grafts had a dramatic effect upon whole blood clotting for all time points tested: Days 0, 14 and 28. Upon visual inspection, the POC eHeparin graft surface remained relatively clean compared with the POC- coated and bare ePTFE grafts ( Fig. 4A). The POC eHeparin grafts had signi ﬁcantly less blood clot formation compared with ePTFE and POC eePTFE grafts at all time points tested up to 28 days ( p < 0.05). In addition, the POC eHeparin grafts had no signi ﬁcant change in anti-clotting activity over the same time period. In contrast, the POC-coated grafts were not signi ﬁcantly different from ePTFE controls at all time points tested. At the 28-day time point, the POC eHeparin and POC eePTFE samples had blood clot formation that was 15.6 10.2% and 70.6 24.4% of ePTFE controls, respectively. The POC eHeparin ePTFE samples were also evaluated for anti- clotting activity after incubation in 100% platelet poor plasma for 1hat37 C. The POC eHeparin samples pre-incubated in plasma had signi ﬁcantly less whole blood clot formation which was only 4.6 5.7% compared with ePTFE control grafts ( p< 0.05) ( Fig. 5). 3.3.2. Platelet adhesion In addition to whole blood clotting, the grafts were evaluated for platelet adhesion using platelet-rich plasma diluted in platelet suspension buffer ( Fig. 6). Platelet-rich plasma contains clotting factors present in the plasma as well as proteins such as ﬁbrinogen, contained within the a-granules of platelets, which are capable promoting clot formation [32]. There were numerous adherent and spread platelets within a clot on the luminal surfaces of both POC- coated and ePTFE control grafts as visualized by SEM ( Fig. 6A and B). The adherent platelets as seen by SEM are approximately 2.5 mm in diameter which corresponds to average platelet size in humans and the adhered and spread platelet morphology is similar to previously reported literature [33,34]. In comparison, there was a dramatic difference with the POC eHeparin grafts which remained relatively pristine with the ePTFE node and ﬁbril architecture still clearly visible ( Fig. 6C). An LDH assay quanti ﬁed the number of adherent platelets on the different vascular graft surfaces. The number of adherent platelets on POC eHeparin ePTFE grafts was signi ﬁcantly less than POC and ePTFE grafts ( p< 0.05). The number of platelets on POC and ePTFE grafts was 4.5 10 7 3.5 10 6and 4.8 10 7 5.8 10 6per cm 2, respectively. In contrast, the number of adherent platelets to POC eHeparin grafts was only 1. 5 10 6 4.7 10 5per cm 2. The relatively small number of adherent platelets on the POC eHeparin surface as visualized by SEM and quanti ﬁed by the LDH assay, demonstrates the ability for the POC eHeparin surfaces to strongly inhibit platelet adhesion when challenged with a relatively high concentration of platelets (platelet- rich plasma).

3.4. Effect of immobilized heparin on vascular cells 3.4.1. HUVEC and BOEC viability and proliferation on POC eHeparin HUVECs and BOECs exhibited good attachment, spreading, and a high degree of viability on all surfaces tested ( Fig. 7). In addition, HUVECs and BOECs stained positive for vWF and VE-Cadherin ( Fig. 8 ). POC eHeparin supported cell proliferation for both endo- thelial cell types although there was some inhibition of HUVECs proliferation on POC eHeparin ( Fig. 9). Speci ﬁcally, the HUVEC surface density after 4 days on POC eHeparin was 1.25 10 5cells/ cm 2compared with 1.63 10 5cells/cm 2and 1.50 10 5cells/cm 2 for TCP and POC surfaces, respectively. Although there were Fig. 4. Bioactivity of POC eHeparin grafts over time. (A) Whole blood clot formation on ePTFE graft segments, POC, and POC eHeparin-coated ePTFE graft segments after 28 days incubation in vitroat 37 C in PBS. Left image panels show graft lumen en faceand right image panels show graft cross-sections. (B) Whole blood clot mass for POC and POC eHeparin-coated ePTFE as percent of ePTFE control surfaces, * p< 0.05, signi ﬁ- cantly less than ePTFE and POC. N 4, mean SD. Note: ePTFE control samples (N 4) were also included for each blood clotting experiment (time points Day 0 eDay 28) and used to normalize whole blood clot mass for POC and POC eHeparin samples. Fig. 5. POCeHeparin coating remains bioactive when exposed to human plasma.

Whole blood clot mass for ePTFE and POC eHeparin grafts pre-incubated with platelet poor plasma as percent of ePTFE control surfaces, * p< 0.05 compared with ePTFE control and ePTFE þplasma samples, n¼ 4, mean SD.

R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 34 Fig. 6.Effect of POC eHeparin on platelet adhesion. SEM micrographs of samples after incubation in platelet-rich plasma: (A) ePTFE, (B) POC-coated ePTFE and (C) POC eHeparin- coated ePTFE. (D) Platelet adhesion quanti ﬁed by LDH, * p< 0.05 compared with ePTFE and POC samples, n 6, mean SD. (A eC) Scale bars: 50 mm. Fig. 7. Cell viability of adherent cells on POC eHeparin. HASMCs, BOECs and HUVECs on TCP, POC and POC eHeparin surfaces after culturing for 4 days. Green: live cells, Red: dead cells. Scale bars: 100 mm. [For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.] R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 35 signiﬁcantly fewer HUVECs on the POC eHeparin surfaces after 4 days, the HUVECs were capable of forming a con ﬂuent cell mono- layer on the POC eHeparin surface after 7 days of culture ( Fig. 9).

Additionally, HUVECs maintained expression of endothelial cell markers and exhibited no signs of decreased cell viability on POC e Heparin. In contrast, BOECs proliferated at the same rate on all surfaces, although at a slower rate when compared to HUVECs. The BOEC surface density after 4 days on POC eHeparin was 1.12 10 4 cells/cm 2compared with 1.15 10 4cells/cm 2and 1.27 10 4cells/ cm 2for TCP and POC surfaces, respectively. The number of BOECs on the different surfaces after 4 days of culture was not statistically signi ﬁcant.

3.4.2. HASMC viability and proliferation on POC eHeparin HASMCs showed good adhesion and viability on all surfaces tested with minimal signs of dead/dying cells ( Fig. 7). The HASMCs on POC and POC eHeparin also had a more elongated and less spread morphology compared with HASMCs cultured on TCP.

Interestingly, HASMCs cultured on POC and POC eHeparin surfaces had greater a-actin expression, which is an indicator of a more physiological contractile phenotype ( Fig. 10B). For HASMC prolif- eration, there were signi ﬁcantly fewer cells on both POC and POC e Heparin surfaces after 4 days of culture ( Fig. 10A). The HASMC surface density after 4 days on POC eHeparin was 7.39 10 4cells/ cm 2compared with 1.32 10 5cells/cm 2and 5.06 10 4cells/cm 2 for TCP and POC surfaces, respectively. The proliferation data is in- line with the a-actin staining, which demonstrates that POC and POC eHeparin surfaces promote a more contractile and less prolif- erative smooth muscle cell phenotype. 3.4.3. Nitric oxide production HUVECs and BOECs cultured on POC and POC eHeparin surfaces produced similar levels of nitric oxide as cells cultured on TCP ( Fig. 11 ). These results demonstrate the ability for POC eHeparin surfaces to support functional nitric oxide producing cells nor- mally present in healthy endothelium.

4. Discussion The quest for the ideal prosthetic vascular graft has generated a signi ﬁcant amount of research on the development of novel biomaterials and surface modi ﬁcation techniques to improve the clinical outcome of bypass surgeries. Of the various strategies investigated over the years, only endothelial cell-based and heparin-based approaches have shown signi ﬁcant promise in regards to patient outcome [35,36]. Nevertheless, more research is needed to overcome the challenges associated with the in vitroor in vivo endothelialization of prosthetic grafts to enable widespread clinical use.

With regard to heparin immobilization, non-covalent and cova- lent strategies have been reported with the latter more desirable to minimize the release of heparin into the systemic circulation [11,37].

Although a comprehensive review of all antithrombogenic strategies for cardiovascular biomaterials is beyond the scope of this current paper, Table 1 provides a summary of heparin-modi ﬁcation strate- gies and current technologies that are speci ﬁc to the development of vascular graft biomaterials. A more detailed review of antith- rombogenic coating technologies for vascular grafts has been prepared by Tatterton et al., and Kapadia et al. [38,39]. In recent Fig. 8. The effect POC eHeparin on endothelial cell phenotype. (A) Immuno ﬂuorescence staining for HUVECs and BOECs, Red: vWF, Green: VE-Cadherin, Blue: cell nuclei (scale bars:

10 0 mm). [For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.]R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 36 years, there has been a growth of commercially available heparin- modiﬁed vascular graft materials such as the Propaten graft (W.L. Gore & Associates, Inc.) and the Intergard graft (Maquet Cardiovascular Inc.). However, the long-term performance and bene ﬁt of such commercially available technologies remains to be answered as the Intergard graft has demonstrated no difference in performance after 5 years compared with human umbilical vein grafts and PTFE grafts [40,41]. Furthermore, the longest prospective and randomized clinical trial to date for the Propaten graft is only for 1 year, but results from this study reveal a decrease in primary graft failure compared with PTFE as a bypass for lower limb ischemia [42].

The Carmeda Bioactive Surface Modi ﬁcation (CBAS ) technique is the basis for the Propaten vascular graft manufactured by W.L.

Gore, which is among the most widely used of commercially available heparin-bonded vascular grafts [7,43]. CBAS is based on layer-by-layer deposition of oppositely charged polyelectrolytes, followed by covalent attachment of heparin, via its reducing end, to polyethyleneimine. The CBAS technology requires toxic chemical reagents and numerous surface modi ﬁcation steps involving nitrous acid-treated heparin and cross-linking of poly- ethyleneimine layers with glutaraldehyde [7,44]. Previous work has shown that biomaterial surfaces prepared with polyethyleneimine by layer-by-layer deposition exhibit cytotoxicity and inhibit cell proliferation [45].

In this study, we developed a new and easily implemented approach to covalently link bioactive heparin to the lumen of ePTFE grafts using a thermally cross-linked POC elastomer and immobili- zation chemistry that targets the carboxyl groups on heparin. As endothelial and smooth muscle cells play an important role in ini- timal hyperplasia and blood vessel homeostasis, the effects of immobilized heparin on these cell types were also evaluated. The POC copolymer chain is composed of a large number of carboxyl and hydroxyl functional groups that are amenable for a variety of surface functionalization strategies for tailoring surface chemistry and/or immobilizing bioactive molecules or peptides. The copolymer can be readily coated onto the nodes and ﬁbrils of ePTFE vascular grafts ( Fig. 3 ) without signi ﬁcantly altering vascular wall thickness, mechanical properties, or ePTFE node/ ﬁbril microstructure [16].

The stability of surface bound molecules for improving vascular conduit blood contacting properties is crucial for long-term vascular graft performance and patency. For example, large amounts of eluted heparin can potentially become lethal due to complications associated with heparin-induced thrombocytopenia [46] . The heparin surface density on POC eHeparin ePTFE grafts ( w 36 e46 ng/mm 2) remained stable over a 28-day period in vitro under physiological conditions with no signi ﬁcant change in heparin surface density over time. In contrast, although heparin- ized stainless steel stents have similarly reported values, they experience considerable degradation (nearly 40%) after one month [33]. Other previous work with heparinization of poly- urethanes has shown a maximum heparin surface density of approximately 11 e23 ng/mm 2with stability evaluated after only 4 days [47e49] .

It is important to evaluate the activity of immobilized heparin because heparin surface density is not necessarily proportional to antithrombogenic activity [50,51]. Aside from factors such as heparin molecular weight and puri ﬁcation methods which may affect heparin activity, the type of covalent modi ﬁcation may alter accessibility of the heparin ’s ATIII-binding site [50]. In this report, POC eHeparin-coated ePTFE grafts signi ﬁcantly inhibited whole blood clotting and maintained long-term bioactivity in vitrofor up to one month. These results are promising because other studies have shown inconsistent immobilized heparin bioactivity after Fig. 9. HUVEC and BOEC cell growth on POC eHeparin. (A) Cell surface density for HUVECs and BOECs on TCP, POC and POC eHeparin surfaces. White bars ¼TCP, gray bars ¼POC, black bars ¼POC eHeparin surfaces for all panels. # p< 0.01 compared with POC, ** p< 0.001 compared with TCP. n 5, mean SD. (B) Con ﬂuent monolayer of HUVECs after 7 days of culture on POC eHeparin (scale bar: 100 mm). R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 37 much shorter time scales (w5 days) [52]. In this regard, the use of spacer molecules such as acrylamide has been shown to improve the bioactivity of immobilized heparin [50]. In this work, the dia- minohexane linker likely allows for improved bioactivity due to increased mobility of heparin ’s ATIII-binding site. Activation of ATIII and subsequent inhibition of pro-clotting factors involved in coagulation are key to reduced blood coagulation when blood comes in contact with surfaces displaying immobilized heparin [53] . The anticoagulant property of immobilized heparin in the grafts was also maintained after incubation with human plasma.

This characteristic is important because clotting factors present in the plasma may adsorb to a blood contacting surface leading acti- vation of the coagulation cascade [54]. It is believed that surfaces with immobilized heparin are capable of inhibiting the process of serum protein adsorption, activation and denaturation involved in thrombus formation [29,55].

Fig. 10. The effect of POC eHeparin on smooth muscle cell growth and phenotype. (A) Cell surface density for HASMCs on TCP, POC and POC eHeparin surfaces. # p< 0.01 compared with POC, ** p< 0.001 compared with TCP. n 5, mean SD. (B) Immuno ﬂuorescence staining for HASMCs, Green: a-actin, Blue: Cell nuclei (scale bars: 100 mm). [For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.] Fig. 11. Nitric oxide production in HUVEC and BOEC cultured on POC eHeparin. NO-positive cells were analyzed by ﬂow cytometry using DAF-2 DA. HUVECs (A) and BOECs (B) cultured on TCP, POC and POC eHeparin surfaces. NO-positive cells were compared relative to “background”samples for which DAF-2 DA cell treatment was omitted.

R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 38 Furthermore, heparin is capable of inhibiting platelet adhesion and activation in the presence of ATIII, but may cause platelet aggregation under certain conditions depending on the molecular weight fraction and concentration [56]. POC eHeparin vascular grafts described herein signi ﬁcantly inhibited platelet adhesion as veri ﬁed by LDH activity and SEM imaging. Additionally, hydrophilic surfaces are associated with improving biocompatibility while inhibiting platelet adhesion and activation [57]. In this regard, the POC eHeparin coating dramatically improved the wettability of the ePTFE vascular graft surface. The covalently attached heparin molecule via the diaminohexane linker may create an ideal hydrophilic layer for further inhibiting platelet adhesion. Although POC has been previously shown to inhibit platelet adhesion, POC cross-linking and rinsing conditions can affect POC surface energy and charge density therefore affecting its interaction with platelets when in contact with blood [16]. Therefore, incorporating heparin into the POC to provide a more robust inhibition of platelet adhe- sion is warranted. One of the reasons for the poor patency of small-caliber ePTFE grafts is due to intimal hyperplasia resulting from the migration and over proliferation of vascular smooth muscle cells [58]. Therefore, the POC eHeparin material was characterized for in vitro compatibility of HASMCs because of the involvement of smooth muscle cell pathology in cardiovascular disease and vascular graft failure. POC and POC eHeparin surfaces were capable of reducing HASMC proliferation and elevating expression of smooth muscle a-actin protein. These results are important because increased HASMC proliferation and reduction in contractile phenotype markers such as a-actin, are implicated in stenosis progression leading to graft failure [59]. Furthermore, heparin signaling and substrate compliance are known to alter HASMC proliferation and phenotype [60,61]. Ourﬁndings are noteworthy in that they are the ﬁ rst to show that poly(diol citrate) elastomers modi ﬁed with heparin are capable of modulating the phenotype of vascular smooth muscle cells in possible combination with heparin signaling and polymer substrate compliance and warrant further investigation regarding the interactions between HASMCs and elastomeric poly(diol citrate) biomaterials. Although previous studies have demonstrated good endothelial cell and BOEC compatibility with unmodi ﬁed POC surfaces, the covalent modi ﬁcation with heparin and its resulting effects on cell behavior must be investigated [13,15]. POCeHeparin surfaces Table 1 Current technologies and methods for developing heparin-modi ﬁed vascular graft biomaterials.

Author/Company Heparin incorporation technique/coating technology Biomaterial Longest performance time point measured Outcome Clinically available Lord et al. [3] Surface adsorption of perlecan heparan sulfate proteoglycan ePTFE vascular grafts 6 weeks in vivo (ovine model) Y platelet adhesion in vitro (fresh samples), improved EC coverage and decreased platelet adhesion in vivo (qualitative analysis only at 6 weeks) Janairo et al. [66]EDC/NHS using diamino-PEG linker Electrospun PLLA vascular graft1 month in vivo (rat model) Heparin-modi ﬁed grafts had 86% patency compared with 43 e57% for control grafts.

Chuang and Masters [49] PEI modi ﬁed polyurethane, aldehyde activated heparin Polyurethane ﬁlms 5 days in vitro [platelet adhesion in vitro (fresh samples), supported EC proliferation (5 days) W.L. Gore & Associates, Inc. Carmeda Bioactive Surface Technology (CBAS ):

layered PEI/dextran sulfate/glutaraldehyde, aldehyde activated heparin [44]Propaten PTFE vascular graft Canine carotid artery interposition model:

3 months. Canine model:

[ graft patency versus ePTFE control grafts and no change in heparin activity [67]. Yes Randomized clinical trial: 1 year for bypass for lower limb ischemia Clinical trial:

Yrisk of primary graft failure by 37% compared with PTFE [42].

Jotec GmbH Flowline Bipore Heparin:

electrostatic bonding interactions/protein substrate PTFE vascular graft Clinical trial comparing femoropoplitea bypass (2004-present) NA European CE Mark Approval only Maquet Cardiovascular Bioline coating: recombinant albumin and covalently attached heparin Fusion Bioline vascular graft, ePTFE and PET (Dacron ) FINEST Phase 3 clinical trial for peripheral artery disease (2011-ongoing) NA European CE Mark Approval only Intervascular Inc. (Acquired by Maquet in 2009) Heparin-bonded collagen coating Intergard composed of PET (Dacron ) vascular grafts Prospective randomized clinical trial: 5 years for above-knee femoropopliteal bypass [40]. No difference in primary patency at 5 years compared with human umbilical vein grafts [40]. Yes Prospective randomized clinical trial: 5 years for above/below-knee femoropopliteal bypass [41].Signi ﬁcantly improved patency at 3 years, but no difference at 5 years compared with PTFE grafts [41].

Perouse Medical Heparin bioactive luminal coating PM Flow Plus Heparin vascular graft, ePTFE NA NAEuropean CE Mark Approval only NA ¼not available; PEI ¼polyethylenimine. Note: Other clinically available heparin-bonded biomaterials include: Dura ﬂo II (Baxter International Inc.), Photolink (SurModics Inc.) and Astute Advanced Heparin Coating (BioInteractions Ltd.) marketed as Trillium Biosurface (Medtronic Inc.) as hemocompatible coating technologies, but are not currently used for vascular grafts. R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 39 supported adhesion, spreading and proliferation of both BOECs and HUVECs. Although the presence of heparin seems to have had an effect on HUVEC proliferation, cells were viable and maintained an endothelial cell phenotype. Nitric oxide secretion is an important endothelial cell mediated process for maintaining a physiologically healthy endothelium and inhibiting thrombus formation. Endo- thelial function was further conﬁrmed by verifying the production of NO. BOECs and HUVECs had comparable NO production when cultured on POC eHeparin.

There is a limited amount of information in the literature regarding the simultaneous characterization of vascular graft surfaces enhanced with antithrombogenic activity and the resulting in ﬂuence on endothelialization and smooth muscle cell function. It is also well known that novel biomaterials used in vascular grafts may also adversely affect endothelial cell function [62]. Moreover, EPC seeding strategies for improving vascular graft thromboresist- ance have heavily relied on the incorporation of collagen, ﬁbrin and ﬁ bronectin for improving cell compatibility [63e65] . However, these extracellular matrix and plasma proteins also promote platelet adhesion and thrombus formation and a subcon ﬂuent or denuded endothelialized surface may provide nucleation sites for thrombus formation. Therefore, the development of POC eHeparin as a multi- functional biomaterial is a signi ﬁcant step towards improving vascular graft performance since it is capable of inhibiting platelet adhesion, blood coagulation and vascular smooth muscle cell growth while simultaneously supporting endothelialization.

5. Conclusion In this report we describe a new approach to impart heparin- mediated thromboresistance and vascular cell compatibility to vascular grafts. The POC eHeparin-coated vascular grafts remained bioactive and signi ﬁcantly inhibited whole blood clotting and platelet adhesion. POC eHeparin supported BOEC proliferation and expression of endothelial cell-speci ﬁc phenotype markers and the production of nitric oxide. Furthermore, POC eHeparin modulated HASMC phenotype via elevated contractile protein expression and decreased cell proliferation rate. Our results support the feasibility of using BOECs and mature endothelial cell types for ex vivoorin situ endothelialization strategies. Due to the ease of synthesis and fabrication, the strategy described herein can be readily adopted to modify other types of devices such as stents, heart valve replace- ments devices and hemodialysis tubing.

References [1] Curi MA, Skelly CL, Meyerson SL, Woo DH, Desai TR, McKinsey JF, et al. Conduit choice for above-knee femoropopliteal bypass grafting in patients with limb- threatening ischemia. Ann Vasc Surg 2002;16(1):95 e101.

[2] Albers M, Battistella VM, Romiti M, Rodrigues AA, Pereira CA. Meta-analysis of polytetra ﬂuoroethylene bypass grafts to infrapopliteal arteries. J Vasc Surg 2003;37(6):1263 e9.

[3] Lord MS, Yu W, Cheng B, Simmons A, Poole-Warren L, Whitelock JM. The modulation of platelet and endothelial cell adhesion to vascular graft mate- rials by perlecan. Biomaterials 2009;30(28):4898 e906.

[4] Wissink MJ, Beernink R, Pieper JS, Poot AA, Engbers GH, Beugeling T, et al.

Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation. Biomaterials 2001;22(2):151 e63.

[5] Chandy T, Das GS, Wilson RF, Rao GH. Use of plasma glow for surface- engineering biomolecules to enhance blood compatibility of Dacron and PTFE vascular prosthesis. Biomaterials 2000;21(7):699 e712.

[6] Zhou Z, Meyerhoff ME. Preparation and characterization of polymeric coatings with combined nitric oxide release and immobilized active heparin. Bioma- terials 2005;26(33):6506 e17.

[7] Larm O, Larsson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modi ﬁed reducing terminal residue. Biomater Med Devices Artif Organs 1983;11(2 e3):161 e73.

[8] Liu L, Guo S, Chang J, Ning C, Dong C, Yan D. Surface modi ﬁcation of poly- caprolactone membrane via layer-by-layer deposition for promoting blood compatibility. J Biomed Mater Res B Appl Biomater 2008;87(1):244 e50. [9] Tseng PY, Rele SS, Sun XL, Chaikof EL. Membrane-mimetic ﬁlms containing thrombomodulin and heparin inhibit tissue factor-induced thrombin gener- ation in a ﬂow model. Biomaterials 2006;27(12):2637 e50.

[10] Sakariassen KS, Joss R, Muggli R, Kuhn H, Tschopp TB, Sage H, et al. Collagen type III induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of ﬁbrin. Arteriosclerosis 1990;10(2):276 e84.

[11] Luong-Van E, Grøndahl L, Chua KN, Leong KW, Nurcombe V, Cool SM. Controlled release of heparin from poly( 3-caprolactone) electrospun ﬁbers.

Biomaterials 2006;27(9):2042 e50.

[12] Allen J, Khan S, Serrano MC, Ameer G. Characterization of porcine circulating progenitor cells: toward a functional endothelium. Tissue Eng Part A 2008; 14(1):183 e94.

[13] Allen JB, Khan S, Lapidos KA, Ameer GA. Toward engineering a human neo- endothelium with circulating progenitor cells. Stem Cells 2010;28(2):318 e28.

[14] Hoshi RA, Behl S, Ameer GA. Nanoporous biodegradable elastomers. Adv Mater 2009;21(2):188 e92.

[15] Motlagh D, Allen J, Hoshi R, Yang J, Lui K, Ameer G. Hemocompatibility evaluation of poly(diol citrate) in vitro for vascular tissue engineering.

J Biomed Mater Res A 2007;82(4):907 e16.

[16] Yang J, Motlagh D, Allen JB, Webb AR, Kibbe MR, Aalami O, et al. Modulating expanded polytetra ﬂuoroethylene vascular graft host response via citric acid- based biodegradable elastomers. Adv Mater 2006;18(12):1493 e8.

[17] Yang J, Motlagh D, Webb AR, Ameer GA. Novel biphasic elastomeric scaffold for small-diameter blood vessel tissue engineering. Tissue Eng 2005;11(11e 12):1876e86.

[18] Yang J, Webb AR, Ameer GA. Novel citric acid-based biodegradable elastomers for tissue engineering. Adv Mater 2004;16(6):511 e6.

[19] Yang J, Webb AR, Pickerill SJ, Hageman G, Ameer GA. Synthesis and evaluation of poly(diol citrate) biodegradable elastomers. Biomaterials 2006;27(9):

1889e98.

[20] Chung EJ, Sugimoto MJ, Ameer GA. The role of hydroxyapatite in citric acid- based nanocomposites: surface characteristics, degradation, and osteoge- nicity in vitro. Acta Biomater 2011;7(11):4057 e63.

[21] Webb AR, Kumar VA, Ameer GA. Biodegradable poly(diol citrate) nano- composite elastomers for soft tissue engineering. J Mater Chem 2007;17(9):

900e6.

[22] Sharma AK, Hota PV, Matoka DJ, Fuller NJ, Jandali D, Thaker H, et al. Urinary bladder smooth muscle regeneration utilizing bone marrow derived mesen- chymal stem cell seeded elastomeric poly(1,8-octanediol-co-citrate) based thinﬁlms. Biomaterials 2010;31(24):6207 e17.

[23] Asahara T, Kawamoto A, Masuda H. Concise review: circulating endothelial progenitor cells for vascular medicine. Stem Cells 2011;29(11):1650 e5.

[24] Ueda M, Alferiev IS, Simons SB, Hebbel RP, Levy RJ, Stachelek SJ. CD47- dependent molecular mechanisms of blood outgrowth endothelial cell attachment on cholesterol-modi ﬁed polyurethane. Biomaterials 2010;31(25):

6394 e9.

[25] Zhu C, Ying D, Mi J, Li L, Zeng W, Hou C, et al. Development of anti- atherosclerotic tissue-engineered blood vessel by A20-regulated endothelial progenitor cells seeding decellularized vascular matrix. Biomaterials 2008; 29(17):2628 e36.

[26] Jantzen AE, Lane WO, Gage SM, Jamiolkowski RM, Haseltine JM, Galinat LJ, et al. Use of autologous blood-derived endothelial progenitor cells at point-of- care to protect against implant thrombosis in a large animal model. Bioma- terials 2011;32(33):8356 e63.

[27] Gupta B, Plummer C, Bisson I, Frey P, Hilborn J. Plasma-induced graft poly- merization of acrylic acid onto poly(ethylene terephthalate) ﬁlms: charac- terization and human smooth muscle cell growth on grafted ﬁlms.

Biomaterials 2002;23(3):863 e71.

[28] Smith PK, Mallia AK, Hermanson GT. Colorimetric method for the assay of heparin content in immobilized heparin preparations. Anal Biochem 1980; 109(2):466 e 73.

[29] Weber N, Wendel HP, Ziemer G. Hemocompatibility of heparin-coated surfaces and the role of selective plasma protein adsorption. Biomaterials 2002;23(2):429 e39.

[30] Grunkemeier JM, Tsai WB, Horbett TA. Hemocompatibility of treated poly- styrene substrates: contact activation, platelet adhesion, and procoagulant activity of adherent platelets. J Biomed Mater Res 1998;41(4):657 e70.

[31] Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, et al.

Detection and imaging of nitric oxide with novel ﬂuorescent indicators: dia- mino ﬂuoresceins. Anal Chem 1998;70(13):2446 e53.

[32] Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg 2006; 118(6):147e e59e.

[33] Yang Z, Wang J, Luo R, Maitz MF, Jing F, Sun H, et al. The covalent immobi- lization of heparin to pulsed-plasma polymeric allylamine ﬁlms on 316L stainless steel and the resulting effects on hemocompatibility. Biomaterials 2010;31(8):2072 e83.

[34] Frojmovic MM, Milton JG. Human platelet size, shape, and related functions in health and disease. Physiol Rev 1982;62(1):185 e261.

[35] Deutsch M, Meinhart J, Zilla P, Howanietz N, Gorlitzer M, Froeschl A, et al.

Long-term experience in autologous in vitro endothelialization of infrain- guinal ePTFE grafts. J Vasc Surg 2009;49(2):352 e62.

[36] Kirkwood ML, Wang GJ, Jackson BM, Golden MA, Fairman RM, Woo EY. Lower limb revascularization for PAD using a heparin-coated PTFE conduit. Vasc Endovascular Surg 2011;45(4):329 e34. R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 40 [37] Heyligers JM, Lisman T, Verhagen HJ, Weeterings C, de Groot PG, Moll FL.A heparin-bonded vascular graft generates no systemic effect on markers of hemostasis activation or detectable heparin-induced thrombocytopenia- associated antibodies in humans. J Vasc Surg 2008;47(2):324 e9. discussion 9.

[38] Tatterton M, Wilshaw SP, Ingham E, Homer-Vanniasinkam S. The use of antithrombotic therapies in reducing synthetic small-diameter vascular graft thrombosis. Vasc Endovascular Surg 2012;46(3):212 e22.

[39] Kapadia MR, Popowich DA, Kibbe MR. Modi ﬁed prosthetic vascular conduits.

Circulation 2008;117(14):1873 e82.

[40] Scharn DM, Dirven M, Barendregt WB, Boll AP, Roelofs D, van der Vliet JA.

Human umbilical vein versus heparin-bonded polyester for femoro-popliteal bypass: 5-year results of a prospective randomized multicentre trial. Eur J Vasc Endovasc Surg 2008;35(1):61 e7.

[41] Devine C, McCollum C. Heparin-bonded Dacron or polytetra ﬂuoroethylene for femoropopliteal bypass: ﬁve-year results of a prospective randomized multicenter clinical trial. J Vasc Surg 2004;40(5):924 e31.

[42] Lindholt JS, Gottschalksen B, Johannesen N, Dueholm D, Ravn H, Christensen ED, et al. The Scandinavian Propaten Triale1-year patency of PTFE vascular prostheses with heparin-bonded luminal surfaces compared to ordinary pure PTFE vascular prostheses ea randomised clinical controlled multi-centre trial. Eur J Vasc Endovasc Surg 2011;41(5):668 e73.

[43] Bosiers M, Deloose K, Verbist J, Schroe H, Lauwers G, Lansink W, et al.

Heparin-bonded expanded polytetra ﬂuoroethylene vascular graft for femo- ropopliteal and femorocrural bypass grafting: 1-year results. J Vasc Surg 2006; 43(2):313 e8. discussion 8 e9.

[44] Lin PH, Bush RL, Yao Q, Lumsden AB, Chen C. Evaluation of platelet deposition and neointimal hyperplasia of heparin-coated small-caliber ePTFE grafts in a canine femoral artery bypass model. J Surg Res 2004;118(1):45 e52.

[45] Brunot C, Ponsonnet L, Lagneau C, Farge P, Picart C, Grosgogeat B. Cytotoxicity of polyethyleneimine (PEI), precursor base layer of polyelectrolyte multilayer ﬁlms. Biomaterials 2007;28(4):632 e40.

[46] Arepally GM, Ortel TL. Heparin-induced thrombocytopenia. N Engl J Med 2006;355(8):809 e17.

[47] Alferiev IS, Connolly JM, Stachelek SJ, Ottey A, Rauova L, Levy RJ. Surface heparinization of polyurethane via bromoalkylation of hard segment nitro- gens. Biomacromolecules 2005;7(1):317 e22.

[48] Bae J-S, Seo E-J, Kang I-K. Synthesis and characterization of heparinized poly- urethanes using plasma glow discharge. Biomaterials 1999;20(6):529 e37.

[49] Chuang T-W, Masters KS. Regulation of polyurethane hemocompatibility and endothelialization by tethered hyaluronic acid oligosaccharides. Biomaterials 2009;30(29):5341 e51.

[50] Michanetzis GP, Katsala N, Missirlis YF. Comparison of haemocompatibility improvement of four polymeric biomaterials by two heparinization tech- niques. Biomaterials 2003;24(4):677 e88.

[51] Lindhout T, Blezer R, Schoen P, Willems GM, Fouache B, Verhoeven M, et al. Antithrombin activity of surface-bound heparin studied under ﬂow condi- tions. J Biomed Mater Res 1995;29(10):1255 e66. [52] Li G, Yang P, Qin W, Maitz MF, Zhou S, Huang N. The effect of coimmobilizing heparin and ﬁbronectin on titanium on hemocompatibility and endotheliali- zation. Biomaterials 2011;32(21):4691 e703.

[53] Sanchez J, Elgue G, Riesenfeld J, Olsson P. Control of contact activation on end- point immobilized heparin: the role of antithrombin and the speciﬁ c antithrombin-binding sequence. J Biomed Mater Res 1995;29(5):655 e61.

[54] Pankowsky DA, Ziats NP, Topham NS, Ratnoff OD, Anderson JM. Morphologic characteristics of adsorbed human plasma proteins on vascular grafts and biomaterials. J Vasc Surg 1990;11(4):599 e606.

[55] Barbucci R, Magnani A. Conformation of human plasma proteins at polymer surfaces: the effectiveness of surface heparinization. Biomaterials 1994; 15(12):955 e62.

[56] Xiao Z, Théroux P. Platelet activation with unfractionated heparin at thera- peutic concentrations and comparisons with a low-molecular-weight heparin and with a direct thrombin inhibitor. Circulation 1998;97(3):251 e6.

[57] Wang Y-X, Robertson JL, Spillman WB, Claus RO. Effects of the chemical structure and the surface properties of polymeric biomaterials on their biocompatibility. Pharm Res 2004;21(8):1362 e73.

[58] Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983;49(2):208 e15.

[59] Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004; 84(3):767 e 801.

[60] Peyton SR, Raub CB, Keschrumrus VP, Putnam AJ. The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells. Biomaterials 2006;27(28):4881 e93.

[61] Letourneur D, Caleb BL, Castellot JJ. Heparin binding, internalization, and metabolism in vascular smooth muscle cells: I. Upregulation of heparin binding correlates with antiproliferative activity. J Cell Physiol 1995;165(3):

676e86.

[62] McGuigan AP, Sefton MV. The in ﬂuence of biomaterials on endothelial cell thrombogenicity. Biomaterials 2007;28(16):2547 e71.

[63] He H, Shirota T, Yasui H, Matsuda T. Canine endothelial progenitor cell-lined hybrid vascular graft with nonthrombogenic potential. J Thorac Cardiovasc Surg 2003;126(2):455 e64.

[64] Schmidt D, Asmis LM, Odermatt B, Kelm J, Breymann C, Gössi M, et al. Engi- neered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors. Ann Thorac Surg 2006;82(4):1465 e71.

[65] Griese DP, Ehsan A, Melo LG, Kong D, Zhang L, Mann MJ, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts. Circulation 2003;108(21):2710 e5.

[66] Janairo RR, Henry JJ, Lee BL, Hashi CK, Derugin N, Lee R, et al. Heparin- modiﬁed small-diameter nano ﬁbrous vascular grafts. IEEE Trans Nano- bioscience 2012;11(1):22 e7.

[67] Begovac PC, Thomson RC, Fisher JL, Hughson A, Gallhagen A. Improvements in GORE-TEX vascular graft performance by Carmeda BioActive surface heparin immobilization. Eur J Vasc Endovasc Surg 2003;25(5):432 e7. R.A. Hoshi et al. / Biomaterials 34 (2013) 30 e41 41