TISSUE ENGINEERING Volume 12, Number 12, 2006 # Mary Ann Liebert, Inc.

Tissue-Engineered Vascular Grafts as In Vitro Blood Vessel Mimics for the Evaluation of Endothelialization of Intravascular Devices
KRISTEN OHALLORAN CARDINAL, B.S.,1 GARRET T. BONNEMA, B.S.,2 HEIDI HOFER,1 JENNIFER KEHLET BARTON, Ph.D.,1,2 and STUART K. WILLIAMS, Ph.D.1

ABSTRACT The accelerating use of minimally invasive procedures for the treatment of cardiovascular disease, and the commensurate development of intravascular devices such as stents, has lead to a high demand for preclinical assessment techniques. A 3-dimensional in vitro blood vessel mimic (BVM) would be ideal for device testing before animal or clinical studies. This is possible based on current capabilities for the creation of tissue-engineered vascular grafts (TEVGs). Using an established method of pressure-sodding human endothelial cells onto a polymer scaffold, a BVM was created in an in vitro bioreactor system under ow. Scanning electron microscopy and immunohistochemistry veried a cellular lining and revealed a luminal monolayer of endothelial cells. After BVM development, bare metal stents were deployed. Stented and unstented BVMs were evaluated using uorescent nuclear staining and optical coherence tomography (OCT). En face and cross-sectional evaluation of bisbenzimide-stained nuclei revealed cellular coverage of the stent surfaces. Cross-sectional evaluation using OCT also illustrated a cellular layer developing over the stent struts. These data support the use of TEVGs as in vitro BVMs for pre-clinical evaluation of the endothelial cell response to stents and endovascular devices.

INTRODUCTION
have become the treatment of choice for many cardiovascular diseases, leading to a high demand for pre-clinical assessments of new intravascular devices. Specically in the area of stents, a wide range of stent materials, coatings, and congurations currently exist, with more variations under development.1 Biodegradable scaffolds, protein coatings, and drug elution are examples of the diversity that has been seen in emerging stent technology.2 4 A great need exists for the preliminary assessment of tissue responses to these newly emerging stent designs and modications, but set-up time and lack of facilities and personnel limit animal studies. Therefore, a 3-dimensional in vitro testing system would allow moreINIMALLY INVASIVE PROCEDURES
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efcient initial assessments to be performed on a large number of coating combinations and stent congurations to direct animal and clinical studies toward the most-promising devices. Over the past several decades, in response to the need for small-diameter vascular bypass conduits, many research groups have developed tissue-engineered vascular grafts (TEVGs).5 These TEVGs are created in a variety of congurations using different material scaffolds and cell types and with different in vitro bioreactor and cultivation systems.6,7 Scaffolds can be synthetic, such as Dacron or expanded polytetrauoroethylene (ePTFE), or biologic, such as elastin or collagen, and can be pre-conditioned or coated in a variety of manners.811 Endothelial cells (ECs) are typically used for the luminal layer of cells. These ECs can

Biomedical Engineering Program, University of Arizona, Tucson, Arizona. Optical Sciences Program, University of Arizona, Tucson, Arizona. 3431

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CARDINAL ET AL. ePTFE.22 To improve cell adhesion to the ePTFE, grafts were pre-conditioned with a 15% FBS solution by capping the distal end of the graft and forcing the solution through the interstitial pores.23

be harvested from large vessels or subcutaneous fat, or they can be derivatives of endothelial progenitor cell lines.1214 Bioreactor systems can vary based on chamber conguration, ow rate, and ow type.15,16 TEVGs are constructed and undergo maturation in an in vitro environment before in vivo implantation. Thus, before implantation, these TEVGs can be thought of as in vitro blood vessel mimics (BVMs). These typically contain at least 2 of the major components of a native blood vessel: the endothelial and sub-endothelial cellular layers. Although in a native environment, there is a more complex intimal, medial, and adventitial structure in addition to blood components, the endothelial and sub-endothelial cellular constituents are key factors in the response to stents and other devices. Specically, the intimal layer of endothelial cells can potentially establish a crucial anti-thrombogenic lining17,18 over the surface of the device after implantation. Promoting or supporting this endothelialization of a stent surface has been the objective of a variety of emerging stent coatings. Thus, an in vitro vessel containing an intimal lining of endothelial cells could be used to evaluate the endothelialization of an implanted stent. In striving toward the goal of creating a tissue-engineered small-diameter graft for peripheral or coronary bypass procedures, our laboratory has created a BVM composed of an ePTFE scaffold with an intimal endothelial cell lining.19 In addition to the potential clinical use of these vessels as TEVGs, we hypothesized that they could be used as in vitro BVMs for the evaluation of intravascular devices, intravascular imaging modalities, drug delivery systems, or pharmaceuticals. Thus, our goal was to determine the feasibility of using this BVM for intravascular device evaluation and intravascular imaging. Specically, the aim of the present work was to assess the potential of our in vitro BVM to evaluate endothelialization of an implanted stent.

Bioreactor system conguration

Each bioreactor system was congured with 2 chambers connected in series by tubing. One of the chambers contained the BVM, and the other served as a media reservoir. The vessel chamber contained 3 ports: a luminal inlet, a luminal outlet, and an extra-luminal outlet. External slide clamps were positioned such that the extra-luminal outlet could be clamped to force luminal ow, or the luminal outlet could be clamped to force transmural ow across the porous ePTFE vessel wall. Both outlets fed into the media reservoir for media recirculation. Tubing was connected to a Watson-Marlow (Cornwall, U.K.) peristaltic pump. The entire system (Fig. 1) was primed with bioreactor medium consisting of M199 supplemented with 10% FBS, 2 mM Lglutamine, 5 mM HEPES buffer, 0.1% Fungizone, and 5% penicillin and streptomycin.

Development of the BVM

Conditioned grafts were aseptically mounted into the vessel chamber, and medium was ushed through the lumen to remove trapped air bubbles. Human microvessel endothelial cells were harvested from T-225 cm2 asks through the application of trypsin. Following deactivation of the trypsin with medium, cells were pelleted and resuspended in bioreactor medium. Cells were pressure sodded onto the lumen of the graft.24 Sodding was performed through the external port with the ePTFE scaffold inside the bioreactor system, and transmural pressure was generated by clamping the luminal ow path. After cell sodding, the bioreactor system was placed on the Watson-Marlow pump in an incubator at 378C with 5% CO2, and transmural pressure was maintained for 1 h. The luminal ow path was then unclamped, and luminal ow was slowly increased to a rate of 15 mL/min. Flow was maintained for 14 days, with medium replaced every third day. This time period allowed for the establishment of a cellular lining on the lumen of the BVMs.

MATERIALS AND METHODS Cell culture and scaffold preparation

Human microvessel endothelial cells, isolated from human liposuction fat, were cultured on 1% gelatin-coated T-225 cm2 asks.20 Cells were cultured at 378C with 5% carbon dioxide (CO2), in M199 supplemented with 10% fetal bovine serum (FBS), 5 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer, 60 mg/mL endothelial cell growth supplement containing heparin,21 and 2 mM L-glutamine. Cells were cultured and expanded to a population sufcient for sodding grafts at a density of 5105 cells/cm2. Cells were used before 5 population doublings. ePTFE, 3 mm and 4 mm inside diameter (Impra Bard, Inc., Tempe, AZ), was used as the BVM scaffold. Graft material was cut into 4.5-cm lengths, mounted on barbed ttings, steam sterilized, and denucleated using a series of graded ethanols to remove air trapped in the interstices of the

Evaluation of the BVM cellular lining

Before device implantation, a set of BVMs was used to verify the establishment of a cellular lining. Vessels were taken out of the bioreactor systems, xed, and cut in half radially. Half of the vessel was then cut again longitudinally. Longitudinal sections were washed with 0.05 M PIPES buffer, dehydrated in a graded series of acetone, and critical-point dried. Dried samples were sputter-coated using a gold target and were observed using a JEOL JSM-820 scanning electron microscope (Tokyo, Japan) to evaluate cell morphology. The other half of the vessels were used for histological and immunohistochemical evaluation. Samples were

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FIG. 1. Bioreactor system conguration showing ow from vessel chamber to reservoir and across pump. External port, expanded polytetrauoroethylene (ePTFE) vessel, and slide clamps are identied. Entire system is placed in incubator with 5% carbon dioxide at and 378C. Multiple bioreactor systems can be supported per pump and multiple pumps per incubator, allowing for large-scale set-up. Color images available online at www.liebertpub.com /ten.

dehydrated, embedded in parafn, and sectioned at 6 mm. Sections were stained using hematoxylin and eosin to verify general histological structure. Specic cellular phenotypes were identied using antibodies against von Willebrand factor (vWF) to identify endothelial cells, a-smooth muscle actin to identify smooth muscle cells, and vimentin to identify undifferentiated cells of mesenchymal origin. Methyl green staining was used to identify background nuclei after immunohistochemical staining had been performed.

balloon was inated to 5 atm, and this pressure was maintained for 30 s to deploy the stent. The balloon was then deated by releasing pressure, and the catheter was removed, leaving the stent in place. Flow was resumed and maintained for an additional 7 to 14 days to allow for a cellular response to the device. One stented BVM was taken down immediately for radiographic evaluation in a Faxitron (Faxitron X-ray, Inc., Wheeling, IL) to illustrate deployment and positioning of the stent inside the vessel.

Stent implantation
Bare metal stents composed of a cobalt-chromium alloy (Guidant Corporation, Santa Clara, CA) were deployed into BVMs after 14 days of BVM development. A subset of BVMs remained un-stented to serve as controls. Stent deployment was performed by inserting a sterile stent-loaded balloon catheter into the bioreactor system through the port upstream from the vessel chamber inlet. Flow was temporarily stopped during the deployment procedure. Insertion and advancement of the catheter was visible through the bioreactor tubing and through the BVM chamber, and thus it was possible to align the loaded stent in the center of the BVM. Upon proper positioning of the catheter and stent, the

Evaluation and quantication of stent cell coverage

After stent deployment and 7 days of ow, stented and un-stented vessels were evaluated for cell coverage. Fixed vessel samples were cut radially and longitudinally to obtain cross-sectional and en face images. Samples were stained with a nuclear specic bisbenzimide (BBI) stain and viewed under ultraviolet epiuorescence to assess cell coverage of the vessel and device surfaces. In stented vessels, endothelialization of the stent-strut luminal surface was quantied using morphometric analysis of uorescent images. En face images from stented BVMs were captured during epiuorescent evaluation and were imported into a computer software program for determination

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CARDINAL ET AL. eosinstained sections veried the general structure of the cellular lining (Fig. 3A), and immunohistochemical staining identied phenotypes of the cells that constituted this cellular lining (Fig. 3). The uid-contacting layer of cells expressed vWF (Fig. 3B), indicating an intimal endothelial cell layer. The sub-endothelial cell layers consisted mostly of vimentin-positive mesenchymal cells (Fig. 3D), although some interspersed smooth muscle cells were noted immediately below the endothelium (Fig. 3C).

of cell density. For each image, strut surface area was traced and quantied based on calibrated measurements, and the number of cell nuclei covering that strut surface was manually counted. Multiple elds of view (n 57) were imaged for each BVM, and the number of cells per mm2 of strut surface area was calculated.

Intravascular imaging of stented BVMs with optical coherence tomography

Optical coherence tomography (OCT) is a high-resolution imaging modality that can be used within the BVM model system to evaluate tissue development and cellular response to stent implantation. OCT was performed intravascularly using a 2-mm-diameter probe-based system described previously.25 Briey, it obtains cross-sectional images by measuring reected near-infrared (1300 nm) light, with a resolution of 16 mm. OCT imaging was performed in stented BVMs immediately after stent deployment and again at 14 days post-deployment. The OCT probe was inserted into the lumen of each BVM, and longitudinal images were acquired at 458 rotational increments.

Feasibility of stent implantation in the BVM system

Stents were successfully deployed in the BVMs because of the access, maneuverability, and visibility within the bioreactor systems. Stenting in each BVM was performed in less than 5 min, allowing minimal disturbance to each system. The radiogram of stent deployment in the BVM (Fig. 4) illustrates that the stent is centered between the barbed ttings that the vessel is mounted on and that the stent is properly deployed in full apposition with the vessel wall.

BBI evaluation of stented BVMs

BBI-stained cross-sectional and en face images of stented BVMs, 7 days post-deployment, are compared with unstented controls in Fig. 5. This type of analysis allowed visualization of the cell nuclei on and around the stent surface. Cross-sectional images of un-stented BVMs (Fig. 5A) illustrated the luminal cell lining, and images of stented BVMs illustrated cellular growth over the stent tynes (Fig. 5B). The en face epiuorescent images of un-stented controls (Fig. 5C) again conrmed the presence of a luminal layer of cells, with conuent cell coverage of the ePTFE vessel. The en face images of stented BVMs illustrated cell coverage of the vessel surface, as well as cell coverage on the stent-strut surface (Fig. 5D). En face images of stented BVMs were used to quantify cell density on the stent-strut surfaces. The frequency distribution of cell densities on stented BVMs is illustrated in Fig. 6. Nineteen images of bare metal stent segments were acquired. The cell density was calculated for each stent segment as described in the Methods section. The total number of segments with each cell density is represented on the bar graph. A majority of stent segments had a density of 50 to 250 cells/mm2. As shown, no segments of the bare metal stents contained fewer than 50 cells/mm2 after 7 days of stent implantation.

RESULTS Establishment of the BVM cellular lining

Scanning electron microscopy evaluation of the BVM luminal surface after 14 days of BVM maturation (Fig. 2) illustrated a conuent layer of cells that exhibited a cobblestone morphology that is consistent with that of an endothelial cell lining. Evaluation of histological sections of un-stented BVMs also veried that a multi-layer cellular lining had developed on the lumen of the ePTFE scaffold. Hematoxylin and

OCT imaging
OCT provided a non-destructive, minimally invasive method for acquiring structural information regarding the presence and thickness of tissue lining the ePTFE scaffold and stent struts. Longitudinal images of a stented BVM immediately post-stenting (Fig. 7A) and 14 days poststenting (Fig. 7B) illustrated the capability of OCT to differentiate between ePTFE scaffold, the luminal lining of

FIG. 2. Scanning electron micrograph of endothelial cell cobblestone morphology on the lumen of the blood vessel mimic.

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FIG. 3. Histologic and immunohistochemical staining of 14 day blood vessel mimics. Hematoxylin and eosin (H&E) staining illustrates the basic structure of the cellular lining (A). Positive von Willebrand factor (vWF) staining was used to identify endothelial cells as the luminal monolayer (B). Alpha-smooth muscle (a-SM) actin antibodies identied smooth muscle cells interspersed directly beneath the endothelial cell lining (C). The majority of cells in the sub-endothelial layers stained positive for vimentin (D). Color images available online at www.liebertpub.com /ten.

cells, stent struts, and cellular growth over the stent struts. After 14 days, the cellular lining had thickened, and cellular growth was documented on and around the stent struts. OCT provided a depth of imaging sufcient to visualize the entire thickness of the BVM and resolution sufcient to visualize these cellular structures of interest.

DISCUSSION
Using tissue-engineered blood vessels, the response of an intimal lining of endothelial cells to device implantation can be evaluated. The work described in this article supports the use of in vitro BVMs to evaluate endothelializa-

tion of implanted stents. This work has shown that a luminal lining of human cells, with endothelial and sub-endothelial layers, is created in the BVM; it is feasible to introduce and properly deploy a stent into the BVM in the bioreactor system; and it is possible to observe and quantify the endothelialization of a bare metal stent. Because of the desire to track the cell response over time, it would be ideal to use a minimally invasive imaging approach, and this work has shown that OCT can be used with the BVM system to monitor the cellular response to stents in a minimally invasive manner. To determine the feasibility of using this system for stent evaluation, it was necessary to establish whether the stent would simply destroy the BVM lining and whether the

FIG. 4.

Radiogram of stent placement. The stent is fully deployed in the blood vessel mimic.

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CARDINAL ET AL.

FIG. 5. Cross-sectional and en face images of bisbenzimide-stained blood vessel mimics (BVMs). Seven days post-deployment, a cellular response to the bare metal stents was observed. Cross-section of un-stented BVMs (A); cross-section of stented BVMs (B); en face image of unstented BVMs (C); and en face image of stented BVMs (D). Multiple images similar to (D) were acquired per sample and were used for quantifying cell coverage. Color images available online at www.liebertpub.com /ten.

tissue would be capable of responding to the device. After 7 and 14 days of implantation, uorescent staining and OCT imaging techniques revealed that the cells were beginning to cover the surface of the bare metal stent. This type of response is consistent with animal studies that show endothelialization of implanted bare metal stents.26 The response would differ based on the specic stent being used;
# Segments with Corresponding Cell Density

7 6 5 4 3 2 1 0 <50 50-150 150-250 251-350 351-450 451-550 Cells/mm2 >550

FIG. 6. Distribution of cell densities on bare metal stents. The number of cells per mm2 of stent strut surface area was calculated from bisbenzimide en face images. The majority of stent segments had coverage between 50 and 250 cells/ mm2. No segments of the bare metal stent struts exhibited less than 50 cells/mm2 after 7 days.

for example, a stent coated with a protein or polymer that promotes endothelial cell adhesion or migration might lead to more-rapid cell coverage of the device. Although this type of evaluation is currently being done in our laboratory, the purpose of this report is to present the model and the cellular response to bare metal stents. Specically in the eld of stent development, the BVM systems are ideally suited for preliminary screening and evaluation of various stent congurations, coatings, and drugelution technologies. In our laboratory, numerous BVMs can be developed simultaneously in an efcient manner that requires less set-up time, fewer personnel, and no animal facilities, unlike with traditional preclinical rabbit or pig models. This allows consistent, large-scale evaluation of intravascular devices, which can elucidate endothelial cell response to stent surfaces before animal studies. Use of the BVM puts devices in contact with human tissue and permits assessment of a large number of coatings and congurations to select the best candidates to progress to traditional preclinical models. Although stents have been the focus of the current work, it would also be possible to use the in vitro BVM model for evaluation of drug-delivery systems, pharmaceuticals, or other intravascular technologies. The primary endpoint in this study was cellular coverage, but evaluation in the BVM can potentially provide information regarding toxicity,

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FIG. 7. Optical coherence tomography images of a stented blood vessel mimic (BVM) immediately after stent deployment (A) and 14 days post-deployment (B). Images show longitudinal cross-sections of expanded polytetrauoroethylene vessel wall (W), cellular lining on BVM lumen (thin arrows), stent struts (brightly reecting sites causing vertical shadows), and cellular growth onto the stent struts after 14 days (fat arrows).

cellular proliferation, migration, and other response mechanisms. Although the BVM model has great potential utility, it is crucial to recognize several key limitations of this system. First, the vessel is based upon a synthetic polymer scaffold that limits the compliance of the vessel wall. This, along with the lack of a true media and adventitia, would make it difcult to evaluate the response of drugs or molecules that target contractility. Also, the system is under low ow and low pressure, which does not identically replicate in vivo conditions. Finally, because of the lack of blood in the system, it would be difcult to fully mimic a native vessel response. In the case of in-stent restenosis, it is thought that several key components originate from circulating cells in the blood,27 and this level of complexity does not currently exist in our model. Despite these limitations, we can consider many different TEVGs that have been created over the past several decades as potential BVM models for other applications. The BVM model discussed in this article is simple and therefore is ideal for initial high-throughput evaluation of endothelial cell response. In addition, just as our model is ideal for evaluating endothelialization of a device surface, other BVMs could serve as ideal mimics for other types of

evaluation. For example, a BVM model that consisted of a more compliant biologic scaffold might be able to provide insight into vasoconstricting or vasodilating pharmaceutical agents. The evaluation of intravascular devices within a BVM has several key advantages. First, it provides a preliminary in vitro testing environment before the use of animals or humans. The BVM offers a more realistic geometry, orientation, and ow condition than in vitro cell culture techniques, which can be especially useful in evaluating different intravascular imaging modalities, drug-delivery systems, or device-deployment methods. For the OCT imaging that was described in this article, it was possible to use the OCT probe intravascularly, just as it would be used in vivo. Also, devices can be tested exactly as they are manufactured and coated. Evaluating a new drug in a cell culture environment may not yield as accurate results as evaluating that drug exactly as it is coated onto a stent platform. Compared with in vitro testing in isolated native vessels, the advantage of BVMs is that they can be developed in a consistent, large-scale manner, which allows more-direct comparisons between devices. Finally, because BVMs can be created with human cells, great potential exists for these in vitro testing systems to elucidate humanspecic responses in a preclinical setting. In conclusion, TEVGs can serve multiple purposes. In addition to the highly researched role as small-diameter bypass conduits, they can also serve as BVMs in an in vitro environment. This article has shown a BVM model that can easily permit stent deployment and possesses an endothelial cell lining that responds to an implanted stent. Thus, this system can provide feedback regarding different stent coatings and congurations and can serve as a model for other newly emerging intravascular technologies.

ACKNOWLEDGMENTS
This material is based upon work supported under a National Science Foundation (NSF) Graduate Research Fellowship (KOC) and National Institutes of Health (NIH) Heart, Lung, and Blood Training Grant HL007955 (GTB). The authors would also like to acknowledge the NSF (Grant 9978820) and NIH (Grant CA109385) for funding the OCT components of this work.

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Address reprint requests to: Stuart K. Williams, Ph.D. Biomedical Engineering Program University of Arizona 1501 N Campbell Ave., LSN 362 Tucson, AZ 857245084 E-mail: skwill@u.arizona.edu