Antithrombogenic Modification of Small-Diameter...

Antithrombogenic Modification of Small-DiameterMicrofibrous Vascular Grafts

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

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

Methods and Results—Microfibrous 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.

Conclusion—Microfibrous 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 being

used 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.1–4

Recently, researchers5–7 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.8–11 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.3,12–14 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.5,15–22 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. [email protected]

© 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 fluoresceinisothiocyanate–conjugated goat anti-rabbit antibody (Jackson Immu-noResearch Inc, West Grove, Pa).

Platelet Adhesion Onto Microfibrous ScaffoldsPlatelet-rich plasma from healthy human volunteers was collected5

and 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 P�0.05.

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 cryopreserved at �20°C.Cryosections were taken at 10-�m thicknesses. Immunohistochem-ical staining was used to analyze the tissue sections with thefollowing primary antibodies: CD31 (BD Biosciences, San Jose,Calif), smooth muscle myosin heavy chain (SM-MHC) (Santa CruzBiotechnology, Santa Cruz, Calif), and CD68 (Santa Cruz Biotech-nology). Immunohistochemistry images were captured with a micro-scope (Zeiss Axioskop 2 MOT).

En Face Immunofluorescence Staining forEndothelial CellsFreshly explanted grafts were transected longitudinally and fixedwith 4% paraformaldehyde for 15 minutes. The samples werewashed with PBS, blocked with 1% BSA, and incubated with mouseanti-rat CD31 antibody and Alexa-Fluor 488 secondary antibody.The samples were washed and mounted onto a slide such that theluminal surface of the graft was in contact with the coverslip.

Mechanical TestingFreshly explanted vascular grafts were cut into 3-mm-wide ringsegments. The tensile strength in the circumferential direction ofthese rings was tested by using a custom-built soft tissue tester. Two0.016-inch-diameter stainless steel rods were placed through thelumen of the ring segment of the graft and attached to polycarbonatecompression grips. The sample was then loaded onto the mechanicaltester, and the applied force and deformation were recorded everysecond via software (Labview v4.0; National Instruments, Austin,Tex). To obtain stress, the cross-sectional area of the sample wasmeasured by histological analysis of adjacent cross sections andcalculated by using the following formula: 2(width of the ring�wallthickness).The elastic modulus was calculated based on the appliedforce, graft deformation, and the dimensions (thickness and width) ofthe rings.25

Results

Direct Fabrication of a Microfibrous TubularGraft With a Microstructure Similar to NativeMatrix FiberBy directly electrospinning polymer fibers onto a rotatingmandrel, we successfully made microfibrous tubular grafts(Figure 1A). This is a significant progress toward makingseamless microfibrous grafts. The PLLA microfibers made byelectrospinning formed a structure (Figure 1B) similar tonative matrix fibers. The average diameter of the fibers wasapproximately 2 �m. As seen in the image, the electrospin-ning process resulted in a highly porous and random structureof fibers.

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Microfibers Could Be Modified by Using PEGand HirudinPEG is capable of creating a brushlike layer onto varioussurfaces and was used to create a protein- and platelet-repulsive surface on the PLLA microfiber scaffold. We firstconjugated PEG onto microfibers and then linked hirudin tothe end of the PEG molecule (Figure 1C). The purpose was touse PEG to resist protein adsorption and platelet adhesion andto use hirudin to inactivate thrombin that reached the luminalsurface of the vascular grafts. The successful conjugation ofPEG and hirudin onto microfibers was confirmed by immu-nostaining for hirudin, which showed the coating of hirudinon individual microfibers (Figure 1D).

PEG and Hirudin-PEG–Conjugated SurfacesReduced the Number and Aggravation ofAdherent PlateletsTo determine whether the grafts modified by PEG andhirudin-PEG can reduce platelet adhesion, platelets wereincubated with untreated grafts and PEG- or hirudin-PEG–modified grafts. As shown in Figure 2A through F, bothCD41 staining for platelets and scanning electron microscopyshowed that microfibrous scaffolds with conjugated PEG andhirudin-PEG had fewer platelets on their surfaces than un-treated microfibrous scaffolds. The reduction of plateletadhesion on PEG and hirudin-PEG microfibrous scaffoldswas statistically significant (Figure 2G), which may helpreduce the possibility of early in vivo graft failures as theresult of thrombosis and platelet adhesion/activation. Thesimilar reduction of platelet adhesion by PEG and hirudin-PEG suggested that PEG contributed to this antiplateletadhesion property.

In addition, scanning electron microscopy demonstratedthe morphological characteristics of the platelets on the

microfibrous PLLA scaffolds. The adherent platelets on theuntreated surfaces appeared to have “spiky” protrusions andpseudopods, indicating that they were activated and aggra-vated by contact with the microfiber sample (Figure 2D). Incontrast, fewer adherent platelets were found on the surfacesmodified by PEG or hirudin-PEG, and these platelets did nothave the same spiky protrusions as seen on the controlsamples (Figure 2E and F).

Figure 1. Structure and chemical modification of microfibrousvascular grafts. A, Scanning electron microscopic (SEM) imageof the electrospun microfibrous vascular graft. The bar indicates0.5 mm. B, SEM of the luminal surface of the electrospun vas-cular graft. The bar indicates 10 �m. C, Conjugation of PEG andhirudin to PLLA. D, Immunostaining of hirudin on nanofibers.The bar indicates 20 �m.

Figure 2. A through F, Representative images of human plate-lets seeded onto PLLA (A and D), PEG-PLLA (B and E), andhirudin-PEG-PLLA (C and F) surfaces. CD41 mouse antihumanantibody and Alexa Fluor 488 secondary antibody were used tostain platelets on PLLA (A), PEG-PLLA (B), and hirudin-PEG-PLLA (C) surfaces. Scanning electron microscopy revealed themorphology and adhesion of platelet on PLLA (D), PEG-PLLA(E), and hirudin-PEG-PLLA (F) surfaces. The bar indicates50 �m in A through C and 5 �m in D through F. G, The numberof platelets bound to each surface. Data are given as themean�SD. *Significant difference (P�0.05, n�4) compared toPLLA.

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PEG- and Hirudin-PEG–Modified GraftsImproved Patency Rates In VivoTo compare the antithrombogenic property of the vasculargrafts in vivo, untreated microfibrous grafts and the graftsmodified with PEG or hirudin-PEG were implanted into thecommon carotid artery of rats for 1 month. Patency wasdetermined by ultrasonography and necropsy. Graft patencywas determined by the unobstructed flow of blood throughthe graft. At 1 month, 6 of 12 (50%) untreated grafts werepatent, 9 of 12 (75%) PEG grafts were patent, and 10 of 12(83%) hirudin-PEG grafts were patent. These results suggestthat both PEG and hirudin improved the patency rate.

To further determine the long-term remodeling of thepatent grafts, hirudin-PEG–modified grafts were implantedfor 6 months. At 6 months, 6 (86%) of the 7 implantedhirudin-PEG grafts were patent. Based on histological anal-ysis, the failed grafts were clogged with thrombus, indicatingthat the imperfect patency rate was related to easiness ofclotting in 1-mm grafts.

The patent grafts from the hirudin-PEG group are shown inFigure 3 as representatives. Figure 3A shows a stereomicro-graph image of an implanted graft moments after the anasto-mosis was completed. The porous structure of the graft wasimmediately filled with red blood cells and other cellularcomponents, and the color of the graft changed from milkywhite to a red. The interrupted suture technique did not resultin any bleeding or leakage at the anastomotic sites. Goodblood flow was observed at both the proximal and distal endsof the graft.

After 1 month, there was visible angiogenesis in the wall ofthe graft (Figure 3B). The presence of newly formed mi-crovessels indicates the integration of the graft into the host’svasculature. In addition, it suggests that the graft is becoming

a living part of the host and that angiogenesis is necessary tosupply nutrients, oxygen, and other diffusible chemicals tothe local cells that reside within and around the graft. Theamount of angiogenesis was slightly less in the 6-monthgrafts (Figure 3C), suggesting that the angiogenesis was partof a short-term wound-healing process in the graft.

A suture site and the characteristics of the graft after 1month were captured by a stereomicroscope (Figure 3D). Theluminal surface was presented by transecting the graft alongthe longitudinal direction. The anastomotic sites were free ofthrombosis and intimal hyperplasia.

Graft patency was also monitored by using Doppler Du-plex (Figure 3E) and MRI (Figure 3F), showing a normalflow rate in the patent grafts.

Endothelialization, Cellular Infiltration,and OrganizationThe patent grafts (either in the untreated group or in thehirudin-treated group) showed similar histological results.Therefore, only hirudin-treated grafts are shown as represen-tatives. Patent grafts (exemplified by hirudin-PEG grafts inFigure 4) showed few signs of thrombosis and/or intimalhyperplasia on the luminal walls of the graft at either the1-month point (Figure 4A) or the 6-month point (Figure 4B).Neotissue formation on the outer surface of the grafts wassignificant at both points. The neotissue in the 1-monthsamples had a highly porous and loose tissue structure on theoutside of the graft (Figure 4C). On the other hand, the6-month sample had dense neotissue with extracellular matrixalignment in the circumferential direction (Figure 4D). Thenucleus staining revealed that there were cells within thewalls of the graft and the neotissue in the outer layer (Figure

Figure 3. Images of 1-mm–internaldiameter hirudin-PEG-PLLA grafts invivo. A through C, Images taken immedi-ately after implantation of the graft (A)and 1 month (B) and 6 months (C) afterimplantation of the graft. D, Stereomicro-graph image of the luminal surface of afreshly explanted 1-month sample. Arrowindicates the suture. E, Duplex Dopplerof the graft taken before euthanatizingthe animal at 6 months. Arrow indicatesthe graft. F, The MRI shows symmetricalblood flow in the control commoncarotid artery (top) and the 1-mm–inter-nal diameter graft (bottom; indicated byan arrow).




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4E and F), suggesting that the graft is capable of supportingcellular ingrowth.

Endothelialization on the luminal surface is important tomaintain the long-term patency of vascular grafts. All patentsamples had complete endothelial coverage at the 1-monthtime point (Figure 5A–C) and the 6-month time (Figure 5D).The newly formed capillaries and microvessels were evidentin the neotissue of the graft (Figure 5A, C, and D), whichsuggests the remodeling of the grafts. Immunofluorescent enface staining revealed that endothelial cells (ECs) aligned inthe flow direction and had a morphological appearancesimilar to ECs in native arteries (Figure 5B).

Smooth muscle cell (SMC) presence and organization arealso important for the long-term stability of vascular grafts.Interestingly, SMC staining showed that SMCs were mostlyin the neotissue surrounding the grafts at 1 month (Figure 5E)and 6 months (Figure 5F) after implantation. The 6-monthsample (Figure 4F) had a clearly defined band of SMCs thatare highly organized and aligned in the circumferentialdirection. There was no sign of a neointima or intimalhyperplasia in these patent grafts.

The inflammatory responses to the PLLA vascular graft weremonitored via immunostaining of CD68, a cell surface marker

for monocytes and macrophages (Figure 5G and H). There werefew CD68-positive cells within the walls of the graft, suggestingminimal inflammatory responses to the grafts.

Mechanical Strength Increased After InVivo RemodelingMechanical strength is critical for the long-term stability ofthe grafts. We performed mechanical tests by using rings

Figure 4. A through F, Cross sections of the explanted hirudin-PEG-PLLA grafts at 1 month (A, C, and E) and at 6 months (B,D, and F) after implantation. Hematoxylin staining (A-D) showedthe patency and structure of the grafts. Nucleus staining by4��,6-diamidino-2-phenylindole (E and F) showed the distribu-tion of cells. Arrows indicate the border of the polymer scaf-folds. The bar indicates 250 �m (A and B) and 100 �m (C-F).

Figure 5. A through G, Endothelialization, SMCs, and inflamma-tory cells in hirudin-PEG-PLLA grafts. ECs were stained byusing CD31 antibody (A–D), SMCs were stained by usingsmooth muscle-major histocompatibility complex (SM-MHC)antibody (E and F), and monocytes and macrophages werestained by using CD68 antibody (G and H). A and C, CompleteEC coverage and adventitial angiogenesis seen 1 month afterimplantation. B, En face staining shows complete endothelializa-tion after 1 month. D, Complete endothelialization after 6-monthimplantation. E, Immunostaining for SM-MHC at 1 month. F,Immunostaining for SM-MHC at 6 months. G, CD68 staining at1 month. H, CD68 staining after 6-month implantation. The barindicates 200 �m (A), 50 �m (B), or 100 �m (C–H).




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of the nonimplanted grafts and the explanted grafts (Figure6A). The representative stress-strain curve of explanted graftsat the 1- and 6-month points is shown in Figure 6B. Theelastic modulus of the grafts before implantation was about3.5 MPa (Figure 6C). After 1 month, a slight increase of theelastic modulus (5.5 MPa) of the grafts was observed. After6 months of implantation, the grafts had a significant increaseof elastic modulus (11.1 MPa), suggesting a significantremodeling of the grafts in vivo.

DiscussionIn this study, we developed small-diameter vascular graftswith high patency rates by using chemical modification ofmicrofibers. These modified acellular grafts showed higherpatency rates, great remodeling, and little intimal thickeningfor 1 and 6 months. To fabricate tubular nanofibrous scaf-folds, we developed an electrospinning system with a rotatingmandrel. The rotating mandrel served as the collector of theelectrospun nanofibers, and the internal diameter of thetubular scaffolds is the same as the diameter of the mandrel.In this study, the internal diameter of the grafts was 1 mm,and it is feasible to make tubular grafts with a much smallerdiameter. Compared with previous fabrication methods, suchas solvent casting and particulate leaching, the microfibrousstructure of electrospun tubular scaffolds provides a matrixmore similar to that in native extracellular matrix of bloodvessels.

In vitro assessment of the scaffold revealed several inter-esting points that eventually led to the enhanced in vivovascular patency in both the short- and long-term vasculargrafts. We showed the ability of the PEGylated surface torepel and maintain the quiescent state of the platelets. The

platelets and presumably other serum proteins were unable toattach onto the brushlike layer of the PEG. Both plateletadhesion and aggravation were reduced on the PEG surface,indicating the biological effects of chemical modification.Hirudin-PEG did not decrease platelet adhesion further com-pared with the PEG surface, suggesting that the resistance toplatelet adhesion could be attributed to the PEG layer.Consistently, in vivo studies showed that the grafts modifiedwith PEG and hirudin-PEG significantly improved the pa-tency rate of the grafts. The slightly higher patency rate of thehirudin-PEG grafts (10/12) suggests that hirudin may have anadditional beneficial effect (eg, inhibition of thrombinactivity).

Endothelialization and sufficient mechanical strength are 2critical aspects for maintaining the long-term stability ofvascular grafts. The chemically modified grafts were able toprevent early thrombosis and to allow ECs to form amonolayer so that long-term patency was achieved.

Both histological and en face staining showed excellentendothelialization in our grafts at the 1- and 6-month time points,contributing to long-term patency. The significant increase of themechanical strength of the grafts at 6 months suggests that therewas an increase of matrix synthesis in and surrounding the graftsin vivo. Based on histological analysis, it is likely that theneotissue on the outer surface of the grafts contributedsignificantly to the enhanced mechanical strength. The valuesof elastic modulus for the grafts are still lower than those ofnative arteries in rats (50 MPa).

The microfibrous grafts were integrated well into nativevasculature, supported by the evidence of angiogenesis andSMC recruitment in the outer layer of the graft. The minimalinflammatory response suggests that the PLLA microfibersare biocompatible materials for vascular regeneration. It ispossible that cells could migrate into the grafts from bothinside and outside surfaces.8 For example, ECs on the luminalsurface could be from microvessels outside the graft, thearteries at 2 ends of the graft, and circulating cells (eg,endothelial progenitor cells) in the bloodstream. Additionalwork needs to be performed to investigate the origin of ECs.The organization of the SMCs in the neotissue outside of thegraft is an interesting observation. These SMCs could be fromthe arteries connected to the grafts or from the surroundingtissues. This is in contrast to the previous observation ofSMCs underneath the endothelium in mesenchymal stemcell–seeded nanofibrous grafts.5 It is likely that cell-seededgrafts allow better cell recruitment and infiltration into thegrafts. Alternatively, because those cell-seeded grafts hadfiber alignment in the circumferential direction, the alignmentof fibers in the cellular grafts could facilitate cell infiltrationinto the 3D structure of the scaffold. These possibilities needto be examined in future studies, and the electrospun scaf-folds could be engineered to further increase cell infiltration.

In the case of other synthetic materials, there were reportson the use of hirudin to modify synthetic polyethyleneterephthalate (Dacron) and expanded polytetrafluoroethylenegrafts. For example, a previous study26 showed the successfulconjugation of hirudin onto the Dacron surface, but in vivoperformance was not reported. Interestingly, direct coating ofexpanded polytetrafluoroethylene grafts with hirudin also

Figure 6. Mechanical characterization of the tensile strength ofhirudin-PEG-PLLA vascular grafts. A, Two stainless steel rodswere placed through the lumen of the ring segment of the graft.The sample was then loaded onto the mechanical tester, andthe applied force and deformation were recorded. B, A typicalstress-strain curve of the vascular graft at 1- and 6-monthimplantation. The elastic modulus was calculated from the slopeof the curve and the graft dimensions. C, The elastic modulus ofgrafts without implantation and at 1 and 6 months after implan-tation (n�4). *P�0.05 vs the other groups of samples. Data aregiven as the mean�SD.




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resulted in excellent patency in larger (4-mm-diameter)grafts.27 A potential disadvantage of noncovalent coating isthe lack of control of surface-binding efficiency and lowhirudin retention rate on the surface. It will be useful tocompare the retention of hirudin on the luminal surface of thegrafts with and without conjugation under hemodynamicconditions.

The use of bioabsorbable microfibrous scaffolds for vas-cular grafts makes it possible to regenerate native bloodvessels. The slow degradation rate of biopolymers, such asPLLA, maintains the mechanical strength of the grafts longenough and allows gradual replacement of synthetic scaffoldsby native matrix with time. The microfibrous structureprovides a mechanically stable module until cellular infiltra-tion and scaffold remodeling eventually lead to a tissuelikeproduct capable of self-support. In this study with 6-monthimplantation, no significant degradation of the grafts wasobserved because the half-degradation time of PLLA islonger than a year.

The methods to directly fabricate tubular constructs byusing electrospinning make it possible to scale up the pro-duction of more uniform and better-quality grafts, opposed tothe rolling technique previously used. The in vitro and in vivoanalysis of the functional performance of the microfibrousvascular grafts provides insight into the mechanisms of theremodeling and regeneration of a blood vessel with micro-structure. The ability to create antithrombogenic small-diameter vascular grafts makes it feasible to offer vasculargrafts available off the shelf.

AcknowledgmentsWe thank Benjamin Lee, BS, and Henry Liu, BS, for their help withgraft preparation and histological analysis; and Michael Wendland,PhD, for his help with MRI.

Sources of FundingThis study was supported in part by Training Grant T1–0007 fromthe California Institute for Regenerative Medicine (Dr Hashi);Training Grant GM56847 from National Institute of General Medi-cine Sciences - Initiative for Maximizing Student Development(NIGMS-IMSD) (Mr Janairo); and grants HL 078534 and HL083900 from the National Institutes of Health (Dr Li).

DisclosuresCraig Hashi and Song Li have financial interest in NanoVasc, Inc.

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

Antithrombogenic Modification of Small-Diameter Microfibrous Vascular Grafts

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