Vessel Bioengineering

Circulation JournalOfficial Journal of the Japanese Circulation Societyhttp://www.j-circ.or.jp

Current Arterial Grafts for Vascular Bypass Surgery

Coronary artery disease (CAD) is a leading cause of death or impaired quality of life for millions of individuals in Japan and the USA. Coronary artery bypass grafting surgery, a common treatment for CAD, currently relies on autologous tissue grafts harvested from either arteries such as the internal mammary artery, gastroepiploic artery, and radial artery, or the saphenous vein, with 10-year patency rates ranging from 90% to 50%, respectively.1,2 However, autografts have several disadvantag-es, including the inconvenience of harvesting and preparing the tissue graft as well as insufficient availability in patients with widespread atherosclerotic vascular disease or in those whose vessels have already been harvested for a previous procedure. In the 1980 s, attempts were made to establish cryopreserved allograft veins (CAV) as a bypass substitute. Such allografts have in fact been used in patients without sufficient autologous graft material.3 Given the poor early and late patency rates, however, CAV have not become widely accepted.4

Expanded-polytetrafluoroethylene (ePTFE, Gortex®), poly-ethylene terephthalate (PET, Dacron®), and polyurethanes are the most commonly used synthetic graft materials for vascular

bypass surgery because of the ease in tailoring the mechanical properties of the material and the relative success in applica-tions that require grafts greater than 6 mm in diameter in a high-flow, low-resistance circulation.5 However, small diam-eter (<6 mm) arterial grafts have not yet shown clinical effec-tiveness because of their poor patency rate related to thrombo-sis and stenosis. To address these challenges, tissue engineering techniques have emerged to make biologically active small-diameter arterial grafts.

Tissue Engineering Blood VesselsThe purpose of tissue engineering is to develop alternative materials that integrate with the patient’s native tissue to restore physiologic function.6 Often, this involves the use of synthetic or natural materials termed “scaffolds” to provide a surface for cell growth and new tissue formation followed by degradation of the scaffold at a later point. Therefore, the traditional con-cept of tissue engineering has 3 components: (1) a tissue-induc-ing scaffold material, (2) isolation and use of cells or cell sub-stitutes, and (3) integration of the cells and the scaffold via a seeding technique.7 Once implanted, biological signaling (hu-moral and mechanical) is thought to be an important factor in

Received November 25, 2013; accepted November 26, 2013; released online December 10, 2013Nationwide Children’s Hospital, Columbus, OH (S.T., N.H., T. Sugiura, H.K., C.K.B., T. Shinoka); Yale University School of Medicine,

New Haven, CT (K.A.R.), USAMailing address: Toshiharu Shinoka, MD, PhD, Nationwide Children’s Hospital, 700 Children’s Drive Columbus, OH 43205, USA.

E-mail: [email protected] doi: 10.1253/circj.CJ-13-1440All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Vessel Bioengineering– Development of Small-Diameter Arterial Grafts –

Shuhei Tara, MD, PhD; Kevin A. Rocco; Narutoshi Hibino, MD, PhD; Tadahisa Sugiura, MD, PhD; Hirotsugu Kurobe, MD, PhD; Christopher K. Breuer, MD; Toshiharu Shinoka, MD, PhD

The development of vascular bioengineering has led to a variety of novel treatment strategies for patients with car-diovascular disease. Notably, combining biodegradable scaffolds with autologous cell seeding to create tissue-en-gineered vascular grafts (TEVG) allows for in situ formation of organized neovascular tissue and we have demon-strated the clinical viability of this technique in patients with congenital heart defects. The role of the scaffold is to provide a temporary 3-dimensional structure for cells, but applying TEVG strategy to the arterial system requires scaffolds that can also endure arterial pressure. Both biodegradable synthetic polymers and extracellular matrix-based natural materials can be used to generate arterial scaffolds that satisfy these requirements. Furthermore, the role of specific cell types in tissue remodeling is crucial and as a result many different cell sources, from matured somatic cells to stem cells, are now used in a variety of arterial TEVG techniques. However, despite great progress in the field over the past decade, clinical effectiveness of small-diameter arterial TEVG (<6 mm) has remained elu-sive. To achieve successful translation of this complex multidisciplinary technology to the clinic, active participation of biologists, engineers, and clinicians is required.

Key Words: Cell seeding; Coronary artery disease; Scaffolds; Small-diameter arterial grafts; Tissue-engineered vascular grafts

REVIEW

Advance Publication by-J-STAGE

TARA S et al.

Scaffolds for Arterial GraftsThe role of the scaffold is to provide a temporary 3-dimen-sional structure for cellular attachment, infiltration, and prolif-eration. The materials possess biomimetic properties and are highly porous, thereby facilitating cellular infiltration, stimula-tion of neotissue formation, and integration with native tissue.11 Applying TEVG to an arterial system, scaffolds must endure arterial pressure, and should demonstrate mechanical properties akin to the native artery, namely strength, durability, and com-pliance. Lastly, the materials must be biocompatible so as not to induce an immunological response.12 Both biodegradable polymers and extracellular matrix-based natural materials sat-isfy these requirements for use as the scaffold of arterial TEVG (Figure 1).

the remodeling process of scaffolds.8 These factors are inter-dependent and indispensable to the formation of highly orga-nized neotissue.

In 1986, Weinberg and Bell generated what was widely re-garded as the first tissue-engineered blood vessel substitute, consisting of cultures of bovine endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts embedded in a collagen gel.9 However, this tissue-engineered construct lacked adequate strength and required reinforcement with a Dacron® mesh. In the years since, tissue-engineered vascular grafts (TEVG) have been greatly improved and refined, ultimately reaching clinical application in a 2001 trial that used an autologous venous-de-rived cell-seeded biodegradable polymeric scaffold for pulmo-nary artery angioplasty in a patient undergoing an operation for pulmonary artery stenosis of a single ventricle.10

Figure 1. Flowchart of creating arterial scaffolds. The role of the scaffold is to provide a temporary 3-dimensional structure for cellular attachment, infiltration, and proliferation. Furthermore, arterial scaffolds have to endure arterial pressure, and should demonstrate mechanical properties akin to the native artery. Lastly, these materials must be biocompatible so as not to induce an immunological response. Both biodegradable polymers and ECM-based natural materials are used to satisfy these requirements. CAV, cryopreserved allograft; ECM, extracellular matrix; ePTFE, expanded-polytetrafluoroethylene; PET, polyethylen terephthalate; PGA, poly(glycolic acid); PLA, poly(lactic acid); PCL, poly(ε-caprolactone); PLCL, poly(l-lactide-co-ε-caprolactone); PGS, poly(glycerol sebacate); PU, polyurethanes; SMCs, smooth muscle cells; SIS, small intestine.

Table. Biodegradable Polymers for Tissue-Engineered Vascular Grafts

Polymer Tm TgInitial tensile

strength (Mpa)

Elastic modulus

(GPa)

Elongation at break

(%)

Degradation period

PGA 230 36 890 8.4 30 2–3 weeks

PLA 170 56 900 8.5 25 6–12 months

PCL 60 –60 50 0.3 70 12 weeks

PLCL (LA/CL=75/25) 140 22 500 4.8 70 8–10 weeks

PLCL (LA/CL=50/50) 105 –17 12 0.9 600 4–6 weeks

GPa, gigapascal; Mpa, megapascal; PCL, poly(ε-caprolactone); PLCL, poly(l-lactide-co-ε-caprolactone); PGA, poly(glycolic acid); PLA, poly(lactic acid); Tm, melting temperature; Tg, glass-forming temperature.


Vessel Bioengineering for Arterial Grafts

life-spans is required to fully evaluate complete degradation of these grafts.

The electrospinning technique, which enables the production of nanofiber-based scaffolds, has been proposed as a promis-ing method of fabricating arterial scaffolds (Figure 2) because of its attributions of improved cellular infiltration and endothe-lialization over standard synthetic grafts.17 Electrospun small-diameter scaffolds using biodegradable polymers, including PCL18 and PLCL,19 have shown good surgical and mechanical properties with a high patency rate in an arterial implantation model. Furthermore, electrospun nanofibers possess the poten-tial of encapsulation and controlled release of drugs,19,20 which may enable a cell-free TEVG.

Extracellular Matrix-Based Natural MaterialsBiologic scaffolds composed of naturally occurring extracel-lular matrix (ECM) have received significant attention for their potential therapeutic application with reduced foreign body response. As previously mentioned, Weinberg and Bell con-structed the first TEVG using a collagen gel as a natural-mate-rial scaffold for neovessel growth. However, the graft lacked sufficient strength and was unsuitable for implantation.9 This construct was evaluated in vivo as an arterial implant only after reinforcement with Dacron®.21 Various methods of improving the mechanical properties of collagen gels (eg, crosslinking agents such as glutaraldehyde) have been investigated, but none has proven to yield a structurally stable TEVG.22 As an alternative to collagen for natural ECM-based scaffolds, fibrin holds particular promise because of its ability to induce colla-gen and elastin synthesis and improved mechanical proper-ties.23 Furthermore, by combining fibrin gels with biodegrad-able polymeric scaffolds followed by seeding of autologous arterial-derived cells,24 endothelialized vessels have been suc-cessfully implanted in the carotid arteries of sheep.25

Decellularized tissue, often in the form of a xenogeneic, can serve as a naturally available scaffold. Decellularization is typ-ically accomplished by treating tissues with a combination of detergents, enzyme inhibitors, and buffers. Decellularized tis-

Biodegradable PolymersBiodegradable polymers serve as temporary scaffolds for blood vessels before being replaced by ingrowing tissue. These poly-mers degrade via hydrolytic cleavage at the ester bond, pro-ducing fragments of diminishing molecular weight. Degrada-tion of these materials is evidenced first by a loss of mechanical properties, followed by a decrease in mass/volume. The rate of degradation of the polymers depends on initial molecular weight, exposed surface area, and crystallinity. The selection of an appropriate material for the scaffold is dependent on a variety of factors such as biocompatibility, mechanical proper-ties, and biodegradability (Table), and this is a crucial first step in designing the constructs for vascular engineering.

Several biodegradable polymers have been investigated for their suitability in arterial tissue engineering applications. Poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL) are commonly used to construct arterial scaffolds because of their history of successful clinical usage.13 Both PLA and PCL have hydrophobic properties and hence are maintained for a long period in the body. Combining these materials with additional synthetic polymers to create copolymers such as poly(l-lactide-co-ε-caprolactone) (PLCL) allows for the fine tuning of mechanical properties and degradation rates through con-trolling the composition ratios and molecular weights (Table). We have confirmed the in vivo feasibility of PLA-PLCL scaf-folds of small-diameter arterial conduits in a high-pressure environment.14

Although slow degradation of some polymers enables the graft to endure high pressure for long periods by retaining its mechanical properties, it also results in delayed tissue remod-eling. Wu et al demonstrated rapid remodeling using a graft made of poly(glycerol sebacate), a fast-degrading elastomer, in a rat arterial implantation model having good patency. Natural proteins such as silk fibroin15 and chitosan16 have also been used as biodegradable materials for small-diameter arterial grafts, and showed long-term patency with favorable vessel remodel-ing. These materials have clear potential for small artery grafts, but further investigation in large animals with longer

Figure 2. Representative electronic microscope images of poly(glycolic acid) (PGA) mesh fiber coated with poly(l-lactide-co-ε-caprolactone) (PLCL) and electrospun poly(lactic acid) (PLA) nanofiber. Electrospinning technique enables the production of nanofiber-based scaffolds, which improves cellular infiltration and endothelialization over standard synthetic grafts.


TARA S et al.

ized, and finally seeded with autologous EPCs or ECs on the graft lumen. This decellularized tissue functioned well as an arterial graft and was gradually remodeled in vivo by host cells.34

Sheet-Based TechniquesSheet-based techniques do not use synthetic or exogenous materials at all; rather, the vessels are created through the use of autologous cells. Fibroblasts and ECs, obtained via biopsy, are cultured on a substrate and lifted off in contiguous layers with preservation of the ECM. These robust cellular sheets are rolled onto the lumen of a tubular scaffold to form a TEVG.35 One clinical study demonstrated the fabrication of 10 TEVG using sheet-based tissue-engineering methodology, followed by implantation as arteriovenous shunts for hemodi-alysis access. Primary patency was maintained in 7 (78%) of the remaining 9 patients at 1 month after implantation and in 5 (60%) of the remaining 8 patients at 6 months after implan-tation.36

Researchers have also reported the development of a novel cell sheet-based technique using temperature-responsive cul-ture surfaces to obtain stable cell sheets of single-cell thick-ness.37 These sheets can be harvested without destruction of the intercellular connections, enabling overlaying onto a second and third cell sheet and subsequent implantation. In our own study using induced pluripotent stem (iPS) cells for TEVG fabrication, this new cell sheet technique improved seeding efficiency dramatically in comparison with traditional static seeding (86.5% vs. 4.9%).38 Based on these features of sheet-based techniques, it may have potential to create a scaffold-free autologous TEVG for arterial circulation.

sue contains intact, structurally organized, and mechanically competent collagen and elastin, but lacks cellular components and DNA,26 and has the advantage of being entirely composed natural ECM, giving numerous advantages in mechanical prop-erties and biocompatibility.27 Recently, many decellularized materials have been commercialized for a variety of therapeu-tic applications. One of the most widely studied of the decel-lularized ECM scaffolds is that derived from the small intes-tinal submucosa (SIS).28 The small-diameter SIS grafts used for replacement of the carotid and femoral arteries exhibited a high patency rate in a canine model,29,30 and had similar me-chanical properties to normal arteries.31

Kaushal et al32 decellularized porcine iliac vessels, seeded them with endothelial progenitor cells (EPCs), and implanted the constructs into ovine carotid arteries. These TEVG con-structs remained patent out to 130 days and were remodeled into neovessel, whereas the unseeded control group occluded within 15 days. These results indicate that decellularized vas-cular scaffolds are susceptible to early failure unless first un-dergoing endothelialization or additional modification. Fur-thermore, elements of the ECM are exposed to physical and chemical stresses during the process of decellularization, which can adversely affect the biomechanical properties of the ECM. This deterioration may ultimately lead to degenerative structural graft failure.33 Additional drawbacks of decellular-ized materials include the inability to modify the ECM content and architecture, variability among donor sources, and risk of viral transmission from animal tissue.

Quint et al have developed a unique method of creating decellularized tissue for small-diameter arterial grafts using biodegradable polymer. Briefly, allogeneic aortic SMCs are cultured on a degradable poly(glycolic acid) (PGA) scaffold in a bioreactor. The engineered vessels are then decellular-

Figure 3. Flowchart of cell sources for tissue-engineered arterial grafts. The use of various cell types in tissue engineering has undergone a remarkable expansion and evolution, and many different cell sources, from mature somatic cells to stem cells, are now used for tissue-engineered arterial graft. BM-MNCs, bone marrow derived mononuclear cells; ECs, endothelial cells; EPCs, endothelial progenitor cells; ESCs, embryonic stem cells; MSCs, mesenchymal stem cells; SMCs, smooth muscle cells.



seeded control group.41 These results highlight the desirability of an EC layer in vascular grafts, but direct EC-seeding of synthetic ePTFE conduits is challenging, costly, and may offer only a slight advantage to the patient.

It is widely accepted that SMCs are an integral component of a stable blood vessel. Throughout development, SMCs are the predominant source of the complex ECM that ultimately defines the mechanical behavior of a vessel.42 Yue et al ex-plored the use of SMCs in TEVG by seeding cultured SMCs onto a biodegradable scaffold and implanting the grafts into rat aortas. The implanted constructs demonstrated rapid neotissue formation in comparison with unseeded controls.43 Niklason et al developed a technique for creating an arterial TEVG by seeding SMCs onto a biodegradable PGA scaffold followed by in vitro culture in a pulsatile radial stress environment for 8 weeks.44 These grafts demonstrated physiologic and mechani-cal functions comparable to native human vessels and are being considered for clinical application.45

Stem Cells and Progenitor CellsBecause matured somatic cells have limited expansion poten-tial, recent studies have explored the use of stem cells or pro-genitor cells for TEVG, including mesenchymal stem cells (MSCs), EPCs, bone marrow-derived mononuclear cells (BM-MNCs), embryonic stem (ES) cells, and iPS cells.

MSCs originate from the mesenchyme, the embryonic con-nective tissue that is derived from the mesoderm. Their ability to differentiate into multiple cell lineages, coupled with their

Cell Sources for Arterial GraftsAdvances in cell biology have been integral to the develop-ment of tissue engineering. ECs and SMCs are the main com-ponents of the intima and media, respectively, of blood vessels. Naturally, these were the first cells used in TEVG investigation and they remain critical to vessel remodeling and repair for successful neotissue formation. As a result of major advances in stem cell technology during the past decade, stem cells have also drawn considerable attention for their potential use in tissue-engineering applications (Figure 3).

Matured Somatic CellsECs synthesize many active substances, such as nitric oxide, fibronectin, heparin sulfate, interleukin-1, tissue plasminogen activator, and various growth promoting factors, to maintain vascular tone, structure, and thromboresistance.39 In 1978, Herring et al introduced a technique in which ECs were har-vested from venous tissue by scraping the luminal surface and then seeding a non-biodegradable prosthetic material, which was incubated prior to implantation in a femoropopliteal ar-tery bypass.40 The presence of a confluent monolayer of ECs on the luminal surface of a vascular graft greatly enhances its thromboresistance and prevents the development of neointi-mal hyperplasia through inhibition of bioactive substances responsible for SMC migration, proliferation, and production of ECM: in 1 study, implantation of EC-seeded ePTFE grafts resulted in significantly better outcomes compared with a non-

Figure 4. Proposed mechanism of neoves-sel formation after implantation of a cell-seed-ed biodegradable scaffold. Early pulse of monocyte chemoattractant protein-1 (MCP-1) and related cytokines from seeded bone mar-row-derived mononuclear cells (BM-MNCs) enhances early monocyte recruitment to the scaffold. Infiltrating monocytes release mul-tiple angiogenic cytokines and growth factors (eg, vascular endothelial growth factor [VEGF]), which recruit smooth muscle cells and endo-thelial cells. Adopted with permission from Roh et al.50


TARA S et al.

scaffold to the clinic, and evidence suggests that this technique is safe and effective to use in pediatric patients undergoing extracardiac total cavopulmonary connection procedures.10,54 Based on the clinical success of TEVG in the venous system, the technique of BM-MNC seeding onto a biodegradable polymeric or decellularized scaffold has been applied to small-diameter arterial grafts, and demonstrated good vessel remod-eling and patency rate.55,56

ES cells are pluripotent cells derived from the inner cell mass of the early embryo. ES cells can proliferate indefinitely while retaining their ability to differentiate into virtually any cell in the human body. They possess greater proliferative capacity than adult stem cells, suggesting that they might be a useful cell source for ex-vivo expansion and seeding. Shen et al demonstrated this concept by seeding ECs derived from mouse ES cells onto a PGA scaffold, and confirming the de-velopment of an EC monolayer.57 Although mouse ES cell research has been ongoing for many years, data on human ES cells are limited. Research on human ES cells is still fairly novel, ES cells having first been discovered in 1998.58 As well as the political and ethical concerns, there are further difficul-ties when ES cells are used as a cell source for TEVG, includ-ing the requirement for homogeneous separation, risks of immunological reaction, untoward genetic changes, and over-all technical difficulty.12

iPS cells can be generated from autologous fibroblasts by introducing 4 factors: Oct3/4, Sox2, c-Myc, and Klf4.59 iPS cells are similar to ES cells in morphology, proliferation, sur-face antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity, but differ with re-spect to epigenetic modification, lifespan, and differentiation potential.60 Importantly, the iPS cell approach offers distinct advantages over ES cell utilization, primarily because iPS cells are autologous and therefore transplantation of cells or engi-neered tissue does not require immunosuppressive therapy. Additionally, iPS cell research obviates the political and ethical dilemma associated with embryo destruction and ES cell har-vest. Studies have yet to demonstrate that iPS cells differentiate into mature vascular cells on the scaffold in vivo. However, iPS cells expanded and differentiated ex vivo may prove to be an effective cell source for constructing TEVG, especially when used in the creation of cell sheets, and it has been pro-posed that seeded iPS cells may actually function in a paracrine manner to induce neovascular formation.38 These develop-ments are promising, but a number of obstacles must still be overcome prior to the implementation of iPS cells in TEVG applications, the most serious being the potential for undiffer-entiated iPS cells to form teratomas following implantation.

Perspectives for the FutureCardiovascular repair currently relies on either autologous blood vessels or synthetic materials; however, cardiovascular disease remains a leading cause of death. Many patients lack suitable donor tissue for autologous grafts and using synthetic grafts results in lower patency rates, risk of infection, and in-ability to grow or remodel. With increasing vascular disease rates, the lack of suitable or superior graft materials is a grow-ing problem. Although the field of vascular tissue engineering has made great progress towards addressing this problem, few patients have benefited from this nascent technology. To be widely accepted, a TEVG should present an option that is less invasive, is cost efficient and time saving, and readily avail-able “off-the-shelf.” Although cell culturing and seeding pro-cesses play an important role in TEVG, conventional methods

presence within multiple tissues of the adult human body, have made MSCs the subject of intense research within the field of tissue engineering.12 The use of MSCs in clinical applications requires an understanding of their unique biological character-istics that contribute to the desired therapeutic effect. The fol-lowing 4 properties are considered to be the most important: (1) ability to migrate to sites of inflammation when adminis-trated systemically, (2) ability to differentiate into various cell types, (3) ability to secrete multiple bioactive molecules ca-pable of inhibiting inflammation and stimulating cell recovery from injury, and (4) lack of immunogenicity coupled with the ability to perform immunomodulatory functions.46 Hashi et al conducted a 60-day in vivo study that demonstrated the anti-thrombogenic property of MSCs with excellent long-term pa-tency and well-organized layers of ECs and SMCs, when seeded onto nanofibrous arterial grafts.47 Because thrombosis is a major problem, as it causes acute occlusion after implanta-tion of arterial graft, this antithrombogenic property opened a new possibility of using MSCs for TEVG.

In 1997, Asahara et al confirmed CD34 positivity on EPCs isolated from adult human peripheral blood.48 Kaushal et al demonstrated that ex-vivo expanded EPCs isolated from the peripheral blood of sheep effectively achieved luminal cover-age after seeding onto decellularized xenogeneic arterial grafts, indicating EPCs possess the ability to endothelialize TEVG.32 Allogeneic decellularized grafts seeded with EPCs have also provided a biological vascular graft that resists both clotting and intimal hyperplasia.34 More recently, an EPC-seeded bio-degradable small-diameter arterial scaffold was used in a canine model, and demonstrated favorable mechanical and biological functional properties.16 Ultimately, the primary advantages of EPCs include medical regulatory feasibility (straightforward isolation via non-invasive sampling of peripheral blood), as well as their ability to home to sites of neovascularization where they serve as potent mediators of vasculogenesis.49

Bone marrow (BM) is an abundant source of stem cells, and BM-MNCs have undergone the most successful translation in human studies of TEVG. One of the advantages of using BM-MNCs for TEVG is that they contain several lineages and differentiated stages of cells, as well as an abundance of cyto-kines that may enhance neovascular development. It was pre-viously believed that the stem cell fraction within the seeded BM-MNC population differentiated into the mature vascular cells of developing neovessel following TEVG implantation. Although we identified small populations of hematopoietic and vascular progenitor cells within the BM-MNC population used for seeding, we observed that the number of seeded cells in the graft decreased rapidly in the first few days after implantation, ultimately resulting in the absence of all BM-MNCs within 1 week post-implantation. As a result of these unique findings, we were the first to suggest that a TEVG transforms into func-tional neovessel in situ via an inflammatory process of vascular remodeling (Figure 4).50 Multiple animal studies have demon-strated that vascular grafts seeded with BM cells may be a reasonable therapeutic option. In 1996, Noishiki et al demon-strated the presence of endothelialization in synthetic vessels created by seeding BM-derived cells onto a biodegradable scaffold.51 Additionally, Matsumura et al reported the results of a study in which scaffolds seeded with BM cells were im-planted as canine inferior vena cava interposition grafts with subsequent development of neovascular tissue.52 Although several in vivo studies showed that BM-MNCs contribute to neovessel development and prevent stenosis,50,53 the precise mechanism remains to be fully elucidated.

We have translated this BM-MNCs seeded biodegradable



19. Wang S, Mo XM, Jiang BJ, Gao CJ, Wang HS, Zhuang YG, et al. Fabrication of small-diameter vascular scaffolds by heparin-bonded P(LLA-CL) composite nanofibers to improve graft patency. Int J Nanomed 2013; 8: 2131 – 2139.

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26. Dahl SL, Blum JL, Niklason LE. Bioengineered vascular grafts: Can we make them off-the-shelf? Trends Cardiovasc Med 2011; 21: 83 – 89.

27. Schmidt CE, Baier JM. Acellular vascular tissues: Natural biomate-rials for tissue repair and tissue engineering. Biomaterials 2000; 21: 2215 – 2231.

28. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007; 28: 3587 – 3593.

29. Sandusky GE Jr, Badylak SF, Morff RJ, Johnson WD, Lantz G. His-tologic findings after in vivo placement of small intestine submucosal vascular grafts and saphenous vein grafts in the carotid artery in dogs. Am J Pathol 1992; 140: 317 – 324.

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37. Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimen-sional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002; 90: e40 – e48.

38. Hibino N, Duncan DR, Nalbandian A, Yi T, Qyang Y, Shinoka T, et al. Evaluation of the use of an induced puripotent stem cell sheet for the construction of tissue-engineered vascular grafts. J Thorac Car-diovasc Surg 2012; 143: 696 – 703.

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42. Wagenseil JE, Mecham RP. Vascular extracellular matrix and arte-rial mechanics. Physiol Rev 2009; 89: 957 – 989.

43. Yue X, van der Lei B, Schakenraad JM, van Oene GH, Kuit JH,

have several limitations, including risk of contamination and need for a specialized clean room and technique. To address this challenge, we developed an entirely closed filtration sys-tem to remove the burden of specialized and costly facilities.61 In this system, harvested cells are passed across a specialized filter, and transferred to a chamber where vacuum suction draws the cells into the scaffold to produce a TEVG in a safe, easy to use, and reproducible way.

To achieve successful translation of this complex multidis-ciplinary technology to the clinic, vascular tissue engineering requires the active participation of biologists, engineers, and clinicians in a concerted and direct effort.

DisclosuresC.B. and T.S. receive grant support from Gunze Ltd and the Pall Corp. This funding was not used to support the work described here.

S.T. and H.K. were recipients of a Banyu Fellowship from Banyu Life Science Foundation International.

For all other authors, no conflicts to disclose.

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