Remodeling of a Cell-Free Vascular Graft withNanolamellar Intima into a NeovesselZihao Wang,†,‡,¶ Chungeng Liu,§,¶ Yi Xiao,⊥ Xiang Gu,†,‡ Yin Xu,†,‡ Nianguo Dong,§

Shengmin Zhang,†,‡ Qinghua Qin,*,⊥ and Jianglin Wang*,†,‡

†Advanced Biomaterials and Tissue Engineering Center and ‡Department of Biomedical Engineering, College of Life Science andTechnology, Huazhong University of Science and Technology, Wuhan 430074, China§Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,Wuhan 430022, China⊥College of Engineering and Computer Science, Australian National University, Canberra, Australian Capital Territory 2601,Australia

*S Supporting Information

ABSTRACT: Advances in cardiovascular materials havebrought us improved artificial vessels with larger diametersfor reducing adverse responses that drive acute thrombosisand the associated complications. Nonetheless, thechallenge is still considerable when applying thesematerials in small-diameter blood vessels. Here we reportthe biomimetic design of an acellular small-diametervascular graft with specifically lamellar nanotopographyon the luminal surface via a modified freeze-cast technique.The experimental findings verify that the well-designednanolamellar structure is able to inhibit the adherence andactivation of platelets, induce oriented growth ofendothelial cells, and eventually remodel a neovessel tomaintain long-term patency in vivo. Furthermore, the results of numerical simulations in physically mimetic conditionsreveal that the regularly lamellar nanopattern can manipulate blood flow to reduce the flow disturbance compared withrandom topography. Our current work not only creates a freeze-cast small-diameter vascular graft that employstopographic architecture to direct the vascular cell fates for revasculature but also rekindles confidence in biophysical cuesfor modulating in situ tissue regeneration.KEYWORDS: small-diameter vascular grafts, nanolamellar structure, freeze-cast, numerical simulation, revascularization

Many vascular surgical procedures, including coronaryartery bypass graft surgery,1 arteriovenous shunts,2

and the treatment of congenital heart disease andpulmonary tracts,3,4 require biologically responsive vascularsubstitutes when autografting, the gold standard, is unavailabledue to the patient’s poor vasculature.5 Constructing vasculargrafts with the properties of antithrombosis and inducibleendothelialization has always been a major challenge but isconsidered a promising method when applied to the treatmentof vascular diseases.6 Until now, although large-diameter (ID >6 mm) vascular grafts have been successfully commercializedand are widely used clinically, no satisfactory products for thereplacement of small-diameter (ID < 4 mm) blood vessels havebeen developed, owing to either graft stenosis in the early stageor intimal hyperplasia in the late stage.7−9

Tissue engineering vascular grafts and preseeding of vascularcells in the scaffolds are currently the primary solutions formicrovascular substitutes, but they often suffer from decline in

the biological activities of implanted cells as well as limitationsof storage and transportation.10−13 To address this issue, inthis work, we have constructed a bioinspired small-diametervascular graft that possesses lamellar nanotopography on theluminal surface to mimic the ridge/groove nanotopography innatural blood vessel via a freeze-cast technique (Figure 1).Compared with the conventional methods of surface topo-graphic engineering, such as photolithography, electron beamlithography, electrostatic spinning, and 3D printing,14−16 thefreeze-cast technique is in favor of bioactive protein/peptide-based biomaterials due to the use of an ice template (Figure1b).Topographic cues of vascular intima play pivotal roles in

tuning acute thrombosis, graft occlusion, and endothelializa-

Received: June 15, 2019Accepted: September 4, 2019Published: September 4, 2019

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© 2019 American Chemical Society 10576 DOI: 10.1021/acsnano.9b04704ACS Nano 2019, 13, 10576−10586

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tion.14,17 The latest studies have shown that platelet adhesionand activation may be dependent on the dimensions of surfacefeatures as well as the aspect ratio of ridge to groove. Highaspect ratio features are incorporated in the design of vascularintima to critically minimize platelet activation and con-sequently prevent graft occlusion.18,19 Accordingly, here weapplied the freeze-cast technique to construct a small-diametervascular graft with a higher aspect ratio on the luminal surfaceto direct cellular behaviors of vascular cells and stimulate self-endothelialization by modulating host cells in situ (Figure 1c).The freeze-cast technique is also a powerful approach to builda high aspect ratio of width to height for interface engineering.Structural integrity and integrated remodeling of artificial

vessels are two central goals in vascular tissue engineer-ing.12,20,21 The former should provide the mechanical strengthof vascular grafts that have the capacity to bear the fluctuantarterial pressure prior to completed endothelialization.22,23 Thelatter is expected to engender excellent biocompatibility andinducible endothelialization of graft composition so that theartificial vessels can be integrated with the surrounding tissueand gradually mediate the replacement and regeneration ofinjured vessels.10,24,25 In accordance with the above require-ments, we designed a vascular graft with blended componentsthat contained (a) the natural materials of silk fibroin andgelatin, with the aim of providing outstanding hemocompat-ibility and elasticity for integrating the graft with adjacenttissues, and (b) the synthetic biodegradable material ofpolycaprolactone (PCL), used as a mechanical sheath to

avoid graft rupture.26−28 In consequence, our study created anacellular small-diameter vascular graft with well-designedluminal lamellar structure and the complementary blendedcomponents to modulate vascular cell behaviors and promoteself-endothelialization.

RESULTS AND DISCUSSIONConsistent with the design criteria, the inner surface of thefreeze-cast vascular grafts exhibited a lamellar structure, andeach lamella was about 10 μm in height and 200 nm inthickness (Figure 2a,c,e,g). The average gap between twolamellas was around 20 μm. The lamellar topography wasoriented along the long axial direction of the vascular grafts.This sharp lamellar nanotopography exhibited a high aspectratio of width to height that fairly reduced the contact area ofplatelets, inhibiting their initial adhesion.18 On the contrary,the control graft fabricated via directly freeze-drying showed anonlamellar and irregular topography (Figure 2b,d,f,h). Bothlamellar and nonlamellar structures were quite stable and couldmaintain their morphologies after 1 month of subcutaneouslyimplantation in vivo (Figure 2i,j). Also, a regularly porousstructure in the middle layer of vascular grafts wassimultaneously produced by the freeze-cast technique, andthis kind of porous morphology could promote the ingrowth ofregenerative tissue compared with the compact middle layergenerated from directly lyophilization (Figure S1). Therefore,the freeze-cast technique using the gradient low temperature isan effective approach to produce such a specially lamellar

Figure 1. General idea of this work. (a) Natural blood vessel shows a regular nanotopography of ridge/groove on the intimal surface. (b)Small-diameter vascular graft with the biomimetic structure is prepared by a freeze-cast technique. Oriented growth of the ice-crystaltemplate guides the formation of a specially lamellar nanotopography into the inner surface of vascular grafts. (c) Cell-free vascular graftwith well-designed nanolamellar structure is used to replace the injured vessel to promote the fast endothelialization and maintain a long-time patency.

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structure in comparison with the ordinary freeze-dryingstrategy.A key challenge of vascular grafts is that they need to

withstand arterial pressure immediately upon implantation.Thus, mechanical strength is crucial for maintaining the graftfunction and integrity prior to complete regeneration of newlyformed vessels.23,29 In this study, we applied PCL, a strongbiodegradable polymer, to prepare an outer sheath on thefreeze-cast vascular grafts by electrostatic spinning (Figure S2).The inner diameter of final vascular graft was about 2.2 mm,and the thickness of graft wall was about 1 mm (Figure 2k).The PCL nanofiber as a mechanical sheath was present in theouter layer of the vascular graft (Figure S2). All results ofstress−strain curve, burst pressure, elastic modulus, andelongation at break showed that the mechanical propertieswere significantly enhanced in comparison with the controlwithout PCL (Figure 2l,m and Figure S3). In vitro degradationfurther confirmed that the vascular grafts with a PCL layermaintained their initial structural integrity, and the ratio ofweight loss was only about 5% after 1 month’s degradation inthe PBS buffer, but the control without PCL coating exhibited

an obvious swelling property and a high ratio of weight loss of20% (Figure 2n). Thus, the outer coating of PCL greatlyimproves the mechanical properties of the vascular grafts, andthis outcome is indispensable to the maintenance of graftintegrity and to the reduction of graft rupture in the earlyphase after implantation.Platelet adhesion and activation are highly associated with

the physicochemical properties of substrate surface.30,31 In thepresent study, the vascular grafts with lamellar nanotopographyexhibited excellent antiadhesion to platelets, and fewer plateletsadhered to the inner surface of the lamellar grafts than that inthe control grafts (Figure 3a,c). Most of adherent platelets onthe lamellar structure maintained a spheroid shape, whereasthe adherent platelets on the nonlamellar surface showedmultiple cellular pseudopods, indicating that the platelets onthe lamellar grafts were less activated.18 The ELISA qualitativeanalysis further demonstrated that the expression of CD62marker was less in the lamellar surface than that in thenonlamellar control (Figure 3d). Also, the aligned lamellarstructure could induce oriented adhesion and growth ofendothelial cells (ECs), while random growth was present in

Figure 2. Material characterization of vascular grafts with and without lamellas. (a, c, e, g) Freeze-cast vascular grafts exhibit a regularlylamellar structure on the inner surface, whereas the directedly freeze-dried grafts show a nonlamellar inner surface (b, d, f, h) (a and b aresample photos, c and d are SEM images, e−h are laser interferometer images). (i, j) HE staining images show that lamellar and nonlamellarstructures of vascular grafts are stable to induce the tissue ingrowth after 1 month of subcutaneous implantation. (k) Inner diameter offreeze-cast grafts is about 2.25 mm and the thickness of graft wall is 1 mm. (l, m) Addition of PCL sheath greatly enhances the mechanicalproperties and (n) decelerates in vitro degradation. Data are expressed as mean ± SD, n = 6, ∗∗P < 0.01.

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the control (Figure 3b). There was no significant difference inthe EC proliferation between the lamellar and nonlamellarsurfaces (Figure 3e). Therefore, the interaction betweenvascular cells and artificial grafts clearly indicated that thelamellar nanotopography effectively inhibited the adhesion andactivation of platelets and significantly promoted the orientedgrowth of ECs, eventually providing a solid foundation forinducing rapid self-endothelialization.Constructing a small-diameter vascular graft with long-term

patency remains a major challenge in vascular tissue engineer-ing due to the generation of thrombosis and intimalhyperplasia.7,32 Endothelialization has been considered as agold standard in the development of vascular grafts, and manydifferent strategies have been suggested to improve endotheli-alization using either preseeding vascular cells or the additionof growth factors and other bioactive molecules.4,33,34 In thisstudy, a special vascular graft with lamellar nanotopography onthe intimal surface was produced to tune cell behaviors of the

vascular cells, promote self-endothelialization, and maintain along-term patency rate. Unlike conventional biochemicalstrategies, this work applies solely topographical cues tostimulate the self-endothelialization of cell-free vascular grafts,indicating that the biophysical cue is a crucial independentfactor capable of modulating the hemocompatibility of artificialvessels. The biophysical cue of nanotopography is also moreconvenient for direct use in vivo as it is rarely influenced by thecomplex environmental factors in the human bodies.35,36

We further explored the possible mechanism of lamellarstructure in inhibiting platelet adhesion and activation. First, itwas obvious that the lamellar topography was capable ofreducing the contact area of platelets to prevent initialadhesion.18,37 Second, previous studies confirmed that bloodflow states within vessels were highly associated with surfacetopological structure that could modulate a series of plateletbehaviors.38,39 The results of numerical simulation demon-strated that the lamellar structure of intimal surface could avail

Figure 3. Interactions between cells and grafts in vitro. (a) Morphological observation of platelets adhesion is shown on lamellar andnonlamellar surfaces. Scanning electron microscopy (SEM) images display that there are fewer platelets on the lamellar surface than that inthe control of nonlamellar surface, and the adhesive platelets present a spheroid or single polar shape on the lamellar surface, whereas thedendritic morphology is activated on the nonlamellar surface (red arrows). (b) Morphological control of the endothelial cells (ECs) ismodulated by substrate topography. The lamellar structure can clearly elongate the ECs and drive them to present oriented adherence andgrowth (red arrow), but random adherence and growth of ECs are shown on the nonlamellar surface. (c) Quantitative analysis of plateletadhesion further confirms that fewer platelets are attached on the lamellar surface, and (d) ELISA results demonstrate that the lamellarnanotopography is favorable to reducing platelet activation. (e) There is no significant difference in the proliferation of ECs betweenlamellar and nonlamellar surfaces. Data are expressed as mean ± SD, n = 6, ∗∗P < 0.01.

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to make blood flow uniformly and reduce disturbed flows(Figure 4 and Videos S1 and S2). As a result, less adhesion andactivation of platelets were observed under the surface withordered lamellas. Therefore, not only could the well-designedlamellar topography directly regulate platelet adhesion byreducing the contact area but also it could affect the blood flowstates in the tubular graft to indirectly manipulate the fate ofplatelets.Luminal surface engineering of artificial vessels plays a

pivotal role in modulating blood cell behaviors and eventually

has a crucial influence on the formation of thrombus.15 Severalprevious works have referred to the surface nanotopographyfor manipulating the hemocompatibility of artificial grafts, butthe intrinsic mechanisms are still unclear.40−42 The currentlyprevalent viewpoint is that the adsorption and accumulation ofblood-related proteins can be modulated by surface phys-icochemical properties and subsequently the type and amountof adherent proteins manipulate blood cell behaviors.6 Here wesuggest another possible mechanism of nanotopography-mediated blood cell behavior based on a vital aspect of

Figure 4. Numerical simulation of blood flow velocity on different inner surface. Two kinds of simulation models were constructed by AnsysR18.2 based on graft surfaces with lamellas and without (w/o) lamellas. The flow velocity indicates a gradient decrease on the lamellarsurface but a sharp decrease on the control due to random obstacles. There is a disturbed flow when blood flows over the obstacles (wherethe red arrow points one of obstacles), whereas the blood goes very smoothly on the lamellar surface because of the guidance of lamellas.The rainbow color bar in the images indicates magnitude of velocity, which increases critically from blue to red.

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hemodynamics. The blood flow is highly associated with fluidshear force that can significantly affect the cell fate of plateletsand blood cells and thus can be used as an indicative marker tomonitor the patency of artificial vessels.43,44 In this study, thevascular grafts with lamellar nanotopography showed excellentpropensity to inhibit the adherence and activation of platelets,and through numerical simulation, we confirmed that the flowvelocity was obviously modulated by the lamellar structure

(Figures 3,4, and S4). Normally, the hemodynamics will besignificantly destructive when the blood flow meets obstacles,and the risk of thrombus growth is obviously elevated by asudden decrease of blood flow velocity. Consequently, thesurface topography of artificial vessels might influence thebehaviors of blood cells by several different pathways involvingthe controls of blood-related protein adsorption andhemodynamics.

Figure 5. In situ monitoring by sonographic images. (a) Native vessel, and (b) vascular grafts with lamellar intima on the carotid arteries ofrabbits at 3 months after implantation (scale bar 2 cm). (c) Real-time blood flow at the operation site of the left carotid artery of the rabbitat 3 months postoperation. (d) Native vessels, vascular grafts with lamellar structure, and vascular grafts without lamellar structure at fourpostimplantation time intervals of 0.5 month, 1 month, 2 months, and 3 months are separately inspected by Doppler ultrasound. Threemonths after implantation, the patency rate of artificial grafts with lamellar structure is very close to that of the native vessel and presentsexcellent hemocompatibility. In contrast, the vascular grafts without lamellar structure exhibit a poor patency rate and cause graft occlusionin the short time of 1 month after implantation. Grafts with lamellas (n = 6), grafts w/o lamellas (n = 6).

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Long-term patency is a primary objective for small-diametervascular grafts.7 Sonographic data confirmed that the vasculargrafts with lamellar structure maintained an excellentantithrombosis feature and facilitated blood flow after 3months of implantation (Figure 5). In contrast, graft occlusionwas detected within the control grafts at 1 month post-operation. Thus, the grafts with lamellar structure cansignificantly enhance the long-term patency rate in comparisonwith the control grafts without lamellar structure (Figure S4).More importantly, at different time intervals of 0.5 month, 1month, 2 months, and 3 months, the flow velocity was quitesimilar between vascular grafts with lamellar structure and

native vessel (Table S1). Thus, these results imply thatendothelialization in the vascular graft with lamellar top-ography could be rapidly formed within a short time afterimplantation.We performed histological analysis of the cross-section and

anastomotic site in longitudinal sections of implanted grafts.The results showed that grafts with lamellas exhibited anoriented round-shaped lumen and a flat inner side viahematoxylin/eosin (HE) staining in cross-section and cellularinfiltration and remodeling at anastomotic site in longitudinalsections (Figure 6). In contrast, the artificial blood vesselswithout lamellar topography were all obstructed by embolisms

Figure 6. Histological evaluation of implanted vascular grafts. (a) Upper panel: optical images of cross sections of native vessel and graftsafter 3 months (scale bar 1 cm). Lower panel: hematoxylin/eosin (HE) staining images (scale bar 500 μm) and their magnified images (scalebar 300 μm). Hematoxylin/eosin (HE) staining shows that the newly formed endothelium clearly presents in the vascular grafts withlamellar intima, but a severe thrombus is generated in the vascular grafts without lamellar structure. (b) Histological assessments of grafts atanastomotic site in longitudinal sections (scale bar 500 and 200 μm). Graft lumen is indicated by ∗. The anastomoses site of graft is in adotted box. (c) Comparison of the vessel lumen area between native vessel and graft with lamellas. (d) Comparison of the wall thickness ofthe vessel among native vessel, graft with lamellas, and graft w/o lamellas. Data are expressed as mean ± SD, n = 3, ∗∗P < 0.01.

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(Figure 6a,b). The comparison of luminal area between nativevessels and grafts with lamellas confirmed the success ofvascular intima formation (Figure 6c). As for wall thickness,both grafts with or w/o lamellas were thicker than native vessel(Figure 6d). Hence, our current findings confirmed that anintegrated cellular layer existed, formed along the inner surfaceof the vascular grafts, indicating the excellent hemocompati-bility of the grafts on guiding host cells to complete rapid self-endothelialization.Verhoeff staining and Masson staining demonstrated that

the lamellar grafts showed oriented distribution of elastin,collagen, and muscle fibers, whereas the control grafts rarelydisplayed a network of elastin fibers and collagen (Figure 7a).Two typical markers of vascular ECs, CD31 and vWF, werepositively stained and concentrated at the borders of vasculargrafts.26 The results indicated that the lamellar intima enabledthe recruiting and remodeling of the endogenous endothelialcells to promote in situ endothelialization of cell-free vasculargrafts (Figure 7a and Figure S5). Positive staining of α-SMAshowed the existence of smooth muscle cells (Figure 7a). Thecomparison of cell number in the remodeled grafts and native

vessels confirmed that the remodeling of cell was done andexcellent (Figure 7b). Protein expressions of α-SMA, CD31,and collagen I were separately measured to assess theregeneration of the vascular inner and media layer usingWestern blot (Figure 7c,d). The results of Western blot andrelative protein level indicated that the grafts with lamellasachieved endogenous cell recruiting and remodeling and ECMdeposition in vivo.

CONCLUSIONSCell-free small-diameter artificial vessels appear essential in theclinical treatment of coronary heart disease and peripheralarterial disease. We currently apply a facile freeze-casttechnique to constructing a small-diameter vascular graft thatemploys the well-designed nanolamellar structure and thebalanced components of natural and synthetic materials. Bothin vitro and in vivo evaluations confirmed the functions offreeze-cast vascular graft on enhancing the hemocompatibility,promoting fast endothelialization, and maintaining a long-timepatency rate. More importantly, we verified that thehemodynamics can be modulated by the surface nanopattern

Figure 7. Characterization of cell remodeling and ECM deposition of vascular grafts. (a) Microscopic images of partial cross-section stainedwith Masson’s trichrome for collagen (blue), Verhoeff’s for elastin (black), and immunofluorescent stained for CD 31 (red), vWF (red), α-SMA (green), and collagen I (red). Cell nuclei were stained with DAPI (blue). Graft lumen is indicated by ∗. (b) Quantitative analysis of cellcounting based on five parts of whole section by DAPI staining (n = 5). (c, d) Protein expressions of a-SMA, CD31, and collagen I wereinvestigated to confirm the formation of intimal and media layer of vascular grafts and their relative protein level. Data are expressed asmean ± SD, n = 3, ∗∗∗p < 0.001.

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of vascular grafts to directly manipulate vascular cell behaviors.Consequently, our current work produces a smart small-diameter vascular graft with a particularly intimal nano-topography to conduct endothelialization in situ and mean-while provides a methodology on designing some nanopatternsto independently remodel vascular regeneration.

METHODSFabrication of Freeze-Cast Vascular Grafts. Silk fibroin was

isolated and purified using our previously reported protocol.45

Vascular grafts were made from the 10% of the regenerated silkfibroin and gelatin solution (5:5) mixed with equal volumes. Thefreeze-cast vascular grafts with lamellar structure were fabricated bythe modified protocol.46 Subsequently, the as-prepared grafts weretreated by methanol and glutaraldehyde and coated with 24% ofpolycaprolactone (PCL) on the external surface based on electrostaticspinning (Voltage, 10 kV). Vascular grafts without lamellar structurewere also prepared as controls by a routine lyophilized method.Material Characterization of Vascular Grafts. Morphological

observation of the vascular grafts was performed by scanning electronmicroscopy (SEM). Mechanical properties of the vascular grafts,namely elastic modulus, burst pressure, tensile strength, andelongation break, were measured using an electronic universal testingmachine. Six samples from each group were tested and all sampleswere soaked in PBS (pH = 7.4) for 10 min at room temperaturebefore testing. For the tensile test, the specimen ends were verticallymounted on the machine with a 100-N load cell, leaving a 10 mmgauge length for mechanical loading. After three preloads, the loadingdeformation data were recorded at a deforming speed of 20 mm/min.The stress−strain curves were calculated from the load deformationdata. The tensile strength, elastic modulus, and elongation at breakwere calculated using a computer program in accordance withaccepted formulas. The burst pressure was performed by means of apressure gauge (Y190, China) and a peristaltic pump (PHDULTRA,Harvard Apparatus, US). In the test, one end of the graft wasconnected to the stainless catheter and wrapped by parafilm and fixedtightly by a steel clamp. The other end of the graft was clamped by asteel clamp. All surfaces touching the grafts were covered with theparafilm to prevent the damage of the grafts and avoid water leakage.The stainless catheter was connected to a pressure gauge and aperistaltic pump. When the graft broke and water leaked from it, theperistaltic pump was started and the number indicated on the pressuregauge recorded. Fourier transform infrared spectroscopy (FTIR) wasused to characterize the variances of material functional groups in thewavenumber range of 400−4000 cm−1 before and after the cross-linked reaction.Degradation Assay in Vitro and in Vivo. In vitro degradation of

lamellar grafts with and without PCL coating was evaluated by PBSbuffer solution (pH = 7.4). Briefly, the samples were first incubated in5 mL of PBS solution at 37 °C, respectively. After one month, sampleswere rinsed in dH2O and lyophilized for the weigh. The degradationratio in vitro was calculated by the weight loss before and afterincubation. For in vivo degradation, the vascular grafts weresubcutaneously implanted in rats to evaluate the lamellas durationin vivo. After one month, samples were explanted and fixed forhistological analysis.In Vitro Evaluation Based on Vascular Cells. Rabbit platelet-

rich plasma (PRP) was first prepared by centrifugation from citratedwhole blood, after which the vascular grafts were incubated with 2 mLof PRP (1 × 108 cells mL−1) for 1 h at 37 °C under shaking condition.The number of adherent platelets was determined by lactatedehydrogenase (LDH). The LDH activity in the lysed plateletsuspensions was measured using an LDH cytotoxicity assay kit(Dojindo Chemical Company, Shanghai, China). Simultaneously,CD62 as an activation marker of platelets was measured to evaluatethe platelet activation. The expression of CD62 was measured byELISA (Qiyi Biological Technology, Shanghai China).Endothelial cells (ECs) were commercially purchased and used to

evaluate cell adhesion and proliferation on different luminal surfaces

of the vascular grafts. To investigate the biocompatibilities of theartificial vessel, cell proliferation on different scaffolds was performedin basal medium (BM) and measured by a Cell Counting Kit-8 assay(CCK-8, Dojindo, Kumamoto, Japan) at the different times of Day 1,Day 3, and Day 5. In brief, 5 × 104 cells were seeded on the artificialblood vessel in 24-well culture plates. The absorbance was measuredat 490 nm on a plate reader (Biotek, USA). Cell adhesion on thescaffolds was observed by laser scanning confocal microscopy (LSCM,Leica, TCS SP5, Germany). Briefly, 5 × 104 cells were seeded on thescaffolds in 24-well culture plates. The adherent cells were separatelyfixed in 4% paraformaldehyde/PBS after 2 days of culture. For LSCM,fixed cells were stained with Actin Tracker Green (Beyotime,Shanghai, China).

Numerical Simulation of Blood Flow Feature. We nextevaluated the effect of geometry on the blood flow features in theintimal topography of the vascular grafts. The numerical simulationswere performed using Ansys R18.2. Two different geometry modelswere built to investigate the velocity profiles through the inner wall ofthe tubes. The models focused (a) locally on small regions of alignedstrips and randomly distributed plaques and (b) globally on thedomains of tubes with walls structured by lamellas. In all models, theinlet and the outlet conditions were specified by a constant velocity(80 cm/s) and zero pressure, respectively. The initial flow directionwas along the x-axis and in the inlet (cross-section of the plate at x =0) the velocity was constant.

In Vivo Evaluation Based on Rabbit Carotid Artery Model.We further evaluated the in vivo performance of the vascular graftsusing a rabbit carotid artery model. Animal studies were carried out incompliance with the protocol approved by the Institutional AnimalCare and Use Committee of HUST. Twelve male mongrel rabbits(1.2−1.8 kg) were randomly divided into two groups of vascular graftswith lamellar structure and the controls. The rabbits were normallyanesthetized by injecting 3−5 mL of 3% pentobarbital sodiumsolution from the auricular vein. After the rabbits became completelyunconscious, we shaved heavy hair around the neck with homemadedepilatory cream, disinfected a region of the neck, and incised theepidermal layer to expose and separate the left internal carotid artery.A segment of the artery (∼2 cm) was transected between clamps, andthen vascular grafts (3 mm in diameter and 2 cm in length) wereimplanted and sewn into the defect position of the rabbit carotidartery. After removal of the clamps, the blood flow was restored, andthe wounds were closed with 3−0 monofilament nylon sutures.Aspirin was administered daily as an anticoagulant for 1 week (2 mg/kg).

Doppler Ultrasound Examination. Doppler sonographic assess-ments of the implanted vascular grafts were performed at thepostimplantation time intervals of 0.5 month, 1 month, 2 months, and3 months. At each time point, the rabbits were anesthetized, and 3Dcolor ultrasonography and real-time flow velocity were collected. Thered and blue colors in the Doppler flow spectrum represent forwardand reverse flow, respectively.

Histological Staining. At the end point of animal evaluation, allrabbits were sacrificed by pentobarbital injection. The conduits wereexplanted and fixed for histological analysis and immunofluorescencestaining based on previously published protocols.

Western Blot. Protein expressions of α-SMA, CD31, and collagenI were separately measured to assess the regeneration of vascular innerand media layers by Western blot based on previously publishedprotocols. The relative protein level was quantified using Image-Jsoftware. Antibodies used are listed in Table S2.

Statistical Analysis. SPSS 22.0 software was used for statisticalanalysis. For mechanical tests, cell proliferation test, and cell adhesion,two independent samples t tests were used to determine thesignificance of differences. Values of p < 0.05, p < 0.01, and p <0.001 were considered to indicate statistically significant (∗), verysignificant (∗∗), and extremely significant (∗∗∗). The results wereexpressed as mean ± standard deviation (SD).

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ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsnano.9b04704.

SEM observation for porous structure of middle layerand nanofiber structure of outer layer, histologicalstaining, elastic modulus and tensile strength, FTIRresults, patency rates, analysis of endothelialization, real-time velocity of blood flow in different time intervals,antibodies used in Western blot (PDF)

Dynamic simulation for blood flow on lamellar surface(MPG)

Dynamic simulation for blood flow on nonlamellarsurface (MPG)

AUTHOR INFORMATIONCorresponding Authors*E-mail: jwang520@hust.edu.cn.*E-mail: qinghua.qin@anu.edu.au.ORCIDQinghua Qin: 0000-0003-0948-784XJianglin Wang: 0000-0002-0733-2097Author Contributions¶Z.W. and C.L. contributed equally.NotesThe authors declare no competing financial interest.

ACKNOWLEDGMENTSThis work was financially supported by the National KeyResearch and Deve lopment Program of China(2017YFC1103900, 2018YFC1105700, 2016YFA0101100),the National Natural Science Foundation of China(31670968, 81601610, 81461148032, 31430029, and31781240266), Sanming Project of Medicine in Shenzhen(SZSM201812055), and the Fundamental Research Funds forthe Central Universities (2019kfyRCPY103).

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