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  • Antithrombogenic Modification of Small-DiameterMicrofibrous Vascular Grafts

    Craig K. Hashi, Nikita Derugin, Randall Raphael R. Janairo, Randall Lee,David Schultz, Jeffrey Lotz, Song Li

    ObjectiveTo develop small-diameter vascular grafts with a microstructure similar to native matrix fibers and withchemically modified microfibers to prevent thrombosis.

    Methods and ResultsMicrofibrous vascular grafts (1-mm internal diameter) were fabricated by electrospinning, andhirudin was conjugated to the poly (L-lactic acid) microfibers through an intermediate linker of poly(ethylene glycol).The modified microfibrous vascular grafts were able to reduce platelet adhesion/aggregation onto microfibrousscaffolds, and immobilized hirudin suppressed thrombin activity that may interact with the scaffolds. This 2-prongedapproach to modify microfibrous vascular graft showed significantly improved patency (from 50% to 83%) andfacilitated endothelialization, and the microfibrous structure of the vascular grafts allowed efficient graft remodeling andintegration, with the improvement of mechanical property (elastic modulus) from 3.5 to 11.1 MPa after 6 months ofimplantation.

    ConclusionMicrofibrous vascular grafts with antithrombogenic microfibers can be used as small-diameter grafts, withexcellent patency and remodeling capability. (Arterioscler Thromb Vasc Biol. 2010;30:1621-1627.)

    Key Words: vascular graft small diameter microfibers biomaterials hirudin

    Arterial replacement is a common treatment for vasculardiseases, with more than 500 000 vascular grafts beingused in the bypass procedures of coronary and peripheralarteries each year. However, small-diameter synthetic vascu-lar grafts frequently have issues with thrombosis and occlu-sion. Several methods have been developed to constructtissue-engineered cellular vessels by using vascular cells.14

    Recently, researchers57 have shown that bone marrow cellscan be used to construct vascular grafts; bone marrowmesenchymal stem cells can resist platelet adhesion and areantithrombogenic in vivo when seeded onto electrospunfibrous scaffolds.5 Because a cellular graft takes days toweeks to construct and special care needs to be taken duringpreservation, shipping, and surgery, in this project, we take analternative approach by fabricating chemically modified acel-lular microfibrous vascular grafts that can be made availableoff the shelf.

    In the past 2 decades, both decellularized native matrix andsynthetic materials have been used to engineer vasculargrafts.811 Synthetic biodegradable polymers, such as poly-(lactic acid), poly(glycolic acid), and their copolymers, havealso been used to make porous vascular grafts by usingtechniques such as solvent casting and particulate leach-

    ing.3,1214 In native blood vessels, collagen and elastin fibersare the major matrix components that provide structural andmechanical support. To simulate the microstructure andnanostructure of native extracellular matrix, researchers haveused an electrospinning technique to fabricate fibrous scaf-folds for vascular graft construction.5,1522 To maintain thepatency of vascular grafts, cell seeding or surface modifica-tion is needed to generate a nonthrombogenic luminal sur-face. Although it has been shown that seeding vascular cellsand bone marrow cells into the grafts can improve the patencyof vascular grafts, whether chemical modification of micro-fibers can generate nonthrombogenic grafts has not beenaddressed. Herein, we engineered the chemical property ofmicrofibers to fabricate acellular vascular grafts that arenonthrombogenic and have long-term patency.

    Hirudin is a polypeptide (65 to 66 amino acids) derivedfrom the saliva of the medicinal leech Hirudo medicinalis. Itis the most potent, naturally occurring, specific inhibitor ofthrombin. In this study, we conjugated hirudin to the poly(L-lactate) (PLLA) microfibers through an intermediate linker ofpoly(ethylene glycol) (PEG). The PEG layer was able toreduce platelet adhesion/aggregation onto microfibrous scaf-folds, and immobilized hirudin could suppress thrombin

    Received on: August 16, 2009; final version accepted on: April 26, 2010.From the University of California, San Francisco and University of California, Berkeley Joint Graduate Group in Bioengineering (C.K.H., R.R.R.J.,

    R.L., and S.L.), University of California, Berkeley; the Department of Bioengineering (C.K.H., R.R.R.J., and S.L.), University of California, Berkeley;the Department of Neurological Surgery (N.D.), University of California, San Francisco; the Department of Medicine and Cardiovascular ResearchInstitute (R.L.), University of California, San Francisco; the Department of Mechanical Engineering (D.S.), University of California, Berkeley; and theDepartment of Orthopaedic Surgery (J.L.), University of California, San Francisco.

    Correspondence to Song Li, PhD, Department of Bioengineering, University of California, B108A Stanley Hall, Berkeley, CA 94720-1762. E-mailsong_li@berkeley.edu

    2010 American Heart Association, Inc.

    Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.110.208348

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  • activity that may interact with the scaffolds. This 2-prongedapproach to modify the microfibrous vascular grafts showedimproved patency and facilitated endothelialization, and themicrofibrous structure allowed efficient graft remodeling andintegration.

    MethodsMicrofibrous Scaffold Fabricationand CharacterizationA 20% weight per volume solution of PLLA (Lactel AbsorbablePolymers, Pelham, Ala), with an inherent viscosity of 1.09 dL/g, wasformulated using 1,1,1,3,3-hexafluoro-2-propanol. The mixture wassonicated for 30 minutes or until all of the PLLA crystals werecompletely dissolved. Electrospinning was performed,5,23,24 with mod-ifications using a novel setup with a rotating stainless steel mandrel(1-mm diameter and 75 rpm) and a spinneret that automaticallymoved back and forth in the longitudinal direction of a mandrel toachieve a uniform thickness of the conduit longitudinally. Thenegative voltage of 4.5 kV was applied to the mandrel, and a positivevoltage of 4 kV was applied to the spinneret by using a high-voltagegenerator (Gamma High Voltage, Ormond Beach, Fla). The electro-spinning process was allowed to proceed until an approximately200-m wall thickness was achieved.

    The conduit was removed from the mandrel and placed into avacuum desiccator for 24 hours to evaporate any residual 1,1,1,3,3-hexafluoro-2-propanol. The quality, thickness, and porosity of themicrofibers were inspected using a scanning electron microscope(Hitachi S-5000). The conduit was trimmed into 7-mm-lengthsegments, sterilized in 70% isopropyl alcohol, placed under germi-cidal UV light for 30 minutes, and washed 3 times with phosphatebuffered saline (PBS).

    Hirudin-PEG ConjugationDi-amino PEG (molecular weight, 3350; Sigma Aldrich, St. Louis,Mo) was covalently linked through the carboxyl groups on the PLLAmicrofibers of the grafts by using the following 0-length cross-linkers: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-ride and N-hydroxysulfosuccinimide (Pierce Biotechnology, Rock-ford, Ill).23 The C-terminus of hirudin (Sigma Aldrich) wascovalently attached to the amine groups on the di-amino PEGmolecules via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-drochloride and N-hydroxysulfosuccinimide. Afterward, the con-duits were incubated with a solution of 100-mg/mL glycine in PBSfor 30 minutes at room temperature to wash away and block anyremaining amine-reactive sites created by cross-linking reagents1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysulfosuccinimide. The conduits were washed with PBS 3times. To verify that hirudin was linked to nanofibers, immobilizedhirudin was stained by using a rabbit antibody against hirudin(American Diagnostica, Inc, Stamford, Conn) and a fluoresceinisothiocyanateconjugated goat anti-rabbit antibody (Jackson Immu-noResearch Inc, West Grove, Pa).

    Platelet Adhesion Onto Microfibrous ScaffoldsPlatelet-rich plasma from healthy human volunteers was collected5and incubated for 30 minutes on the microfibrous scaffolds with orwithout conjugation: PLLA, PEG-PLLA, and hirudin-PEG-PLLA.Samples were processed and analyzed using 2 techniques: scanningelectron microscopy and immunofluorescence staining with 1:100mouse antihuman CD41 antibody (Laboratory Vision, Fremont,Calif). The total number of adherent platelets from these 4 imageswas summed, and the results from multiple experiments from 3individual donors were subjected to ANOVA. A Holm t test wasused to calculate statistical significance for P0.05.

    Animal StudiesAll procedures were approved by the Institutional Review BoardService and the Institutional Animal Care and Use Committee at the

    University of California, San Francisco. Female Sprague-Dawleyrats (weight, 200 to 240 g) were obtained from the Charles Riveranimal facility. The rats were anesthetized with 2.0% isoflurane in70% nitrous oxide and 30% oxygen. The right common carotidartery was dissected, clamped, and transected; and the graft wassutured end to end with 6 to 8 uninterrupted stitches using a 10-0needle. No heparin or any other anticoagulant was used at any pointbefore, during, or after the implantation procedure. For 1-monthstudies, 12 animals were used in each experimental group. For6-month studies, 8 animals were used in each experimental group.After 1 to 6 months, the animals were reanesthetized and the vasculargrafts were resected and washed with heparinized saline to removethe remaining blood.

    Histological AnalysisFor histological analysis, the sample was placed into an optimalcutting temperature (Sigma Aldrich) and cryopres