Review Article
The Current Status of Tissue-Engineered Vascular Grafts Vita N. Jaspan and George L. Hines, MD
Abstract: Tissue-engineered vascular grafts (TEVGs) are currently being developed to overcome the limitations and complications associated with traditional synthetic grafts. This article aims to review the current status of research into the production and use of tissue-engineered grafts. TEVGs have a number of theoretical advantages over synthetic grafts. The results of animal and human studies have been promising, but more work must be done before TEVGs can replace traditional grafts. Key Words: tissue-engineered vascular grafts, sheet-based, scaffold-based, electrospinning (Cardiology in Review 2015;23: 236–239)
V
ascular grafts have been used in humans since the early 1900s to treat a variety of acquired and congenital cardiac and vascular conditions. Uses include creation of cardiac shunts in congenital heart defects, repair of aneurysms, repair of injured blood vessels, bypass of stenotic and occluded blood vessels, creation of hemodialysis access, and as creation of portosystemic shunts. The first known use of vascular grafts in humans was performed by Goyannes who used autologous popliteal vein graft to repair a popliteal aneurysm in 1906.1 Later, in 1948, Kunlin performed the first saphenous vein femoropopliteal bypass surgery.1 By the 1950s, synthetic materials came into use to compensate for the lack of available autologous vascular tissue. At this time, Voorhees, Blakemore, and DeBakey used Dacron grafts to treat aortic aneurysms.1 Smaller size Dacron and polytetrafluoroethylene (PTFE) grafts were then introduced in the 1960s and 1970s, and intravascular stents were first introduced in the 1970s and 1980s.1 Although very effective, prosthetic grafts and stents can result in complications including intimal hyperplasia, thrombosis, and infection, especially in arteries of small diameter. Theoretically, some of these problems could be avoided by using native vessels for grafting. Because autogenous tissue may not be available in sufficient size or quantity for use as vascular grafts, tissue-engineered vascular grafts (TEVGs) have been developed. TEVGs are grown using the patient’s own cells and are therefore, by definition, made from autologous tissue. Because they are native in origin, TEVGs should be able to more closely resemble a patient’s vasculature, including vasoreactivity and biomechanics, thus increasing the probability of long-term patency. TEVGs could also be very effective in pediatric patients, where they have the potential to grow and remodel with the patient. There are multiple approaches to the development of TEVGs, all of which are outgrowths of the first TEVG created by Weinberg and Bell,2 which consisted of bovine endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts, all within a tubular collagen matrix. This graft, unfortunately, was unable to withstand physiologic
From the Division of Vascular Surgery, Department of Thoracic and Cardiovascular Surgery, Winthrop University Hospital, Mineola, NY. The authors declare no conflict of interest in the preparation of this manuscript. Correspondence: George L. Hines, MD, Winthrop Cardiovascular Associates, 120 Mineola Blvd, Suite 300, Mineola NY 11501. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 1061-5377/15/2305-0236 DOI: 10.1097/CRD.0000000000000060
236 | www.cardiologyinreview.com
pressures. More recent approaches vary in the types of cells, types of scaffolds, and in the humoral and mechanical signals involved in the regulation and maintenance of the neotissue.3 The various approaches to the development of TEVGs are shown in Figure 1.
SHEET-BASED TISSUE ENGINEERING OF CELL SELF-ASSEMBLY VESSELS One method of TEVG generation, known as sheet-based engineering, has already been used clinically for the creation of hemodialysis access. In this method, sheets of fibroblasts are extracted from biopsies of the patient’s skin and superficial veins, wrapped around a stainless steel mandrel, and cultured for 10 weeks in a bovine serum. The mandrel is then removed, and the sheets are seeded with ECs to promote nonthrombogenicity.4 Production of these grafts takes between 6 and 9 months. Between 2004 and 2007, McAllister et al5 implanted sheet-based grafts in 10 different patients who had at least one previous access failure and who were considered to be at a high risk for graft failure. Of these, three-fourths were patent after 1 month, and three-fifths were patent after 6 months. Failures were secondary to dilatation and the development of aneurysms, despite the high burst strength and expected low immune response of the grafts. Although an encouraging result, these grafts are very costly, time consuming to produce and must be improved before they can replace traditional synthetic grafts. Wystrychowski et al6 recently implanted three sheet-based allogeneic grafts as brachial-artery arteriovenous shunts for hemodialysis access. This was the first use of allogeneic TEVGs in humans. The grafts were formed from cells extracted from 2 donors and were not endothelialized. After 7–12 weeks, all 3 grafts were punctured and used for hemodialysis access, at which time good blood flow was observed. No immunologic or inflammatory responses were observed. After approximately 4 months, intervention was required in all 3 grafts, primarily to correct stenoses of axillary and central veins. Within 7 months of implantation, all 3 grafts failed for a variety of reasons, including thrombosis, an unrelated infection, and physical injury. Other problems regarding slow-healing after puncture were noted, which the authors suggested were likely influenced by the lack of endothelialization of these grafts. This case study yielded very promising results regarding the potential for widespread TEVG use in humans. No infections or aneurysms developed in any of the grafts, but thrombosis and quick failure of all 3 grafts indicate that more work must be done to improve the durability of allogeneic TEVGs. This study also reveals that ECs may not be necessary to maintain mechanical stability of the graft, as was previously thought.
SCAFFOLD-BASED TECHNOLOGIES Most current strategies for the development of TEVGs involve scaffold-based technologies. In 1978, Herring et al7 proposed seeding ECs onto the surface of synthetic conduits. These grafts showed no advantage over traditional synthetic grafts. This approach was later refined when Mazzucotelli et al8 seeded ECs onto PTFE grafts with fibrinolytically inhibited fibrin glue, which improved patency of small-diameter vascular grafts. This improvement was because of the increased adhesion of cells to the graft. Using this strategy, Deutsch et al9 demonstrated a 9-year patency rate of 65% when compared
Cardiology in Review • Volume 23, Number 5, September/October 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cardiology in Review • Volume 23, Number 5, September/October 2015
The Current Status of TEVGs
FIGURE 1. Brief overview of the various approaches to tissue-engineered vascular graft (TEVG) development. with 16% for the control group who received untreated ePTFE grafts, for infrainguinal reconstructions. These synthetic materials cannot remodel, thus preventing the growth and vasoreactivity, which would increase their utility.3 For this reason, biodegradable synthetic polymer scaffolds are preferred, which degrade over time, leaving behind vessels that more closely resemble native tissue. Niklason et al10 cultured porcine aortic SMCs on a polyglycolic acid (PGA) fabric. PGA is a biodegradable material that begins to degrade after 2–4 weeks and is completely absorbed within 6 months of implantation. Poly-l-lactic acid can be copolymerized with PGA to regulate degradation rates.11 The investigators incubated the seeded scaffold in a pulsatile bioreactor to mimic physiologic conditions, which promoted the production of a robust extracellular matrix (ECM).10 Proper ECM development is vital to the success of the graft—improper development can result in stenosis and dilatation.12 The resulting vessel was vasoreactive and had burst pressures in excess of 2000 mm Hg.10 When Dahl et al13 transplanted a PGA graft seeded with human cells into baboons as arteriovenous conduits, an 88% patency was observed over a 6-month period. No aneurysmal dilatation or calcification was observed, and minimal intimal hyperplasia was evident. After 6 months, much remodeling occurred, and a loose, fibrous adventitia-like layer was visible. Despite promising results, the cell-culture technique is very time-consuming, limiting the utility of these technologies. Furthermore, the process of culturing cells requires the use of serum from nonhuman species, thus increasing the risk of contamination.14 To overcome these problems associated with in vitro cell cultures, Matsumura et al15 found that bone marrow derived mononuclear cells (BM-MNC) can develop into native-like vasculature. These cells are readily available in the iliac crest and can be isolated the day of surgery, thereby obviating the need for cell culture. First, they implanted these BM-MNC seeded grafts into the inferior vena cava (IVC) of dogs. Using immunohistochemistry, they confirmed that the seeded bone marrow cells (BMCs) adhered to the poly-l-lactic acid scaffold, an alternative biodegradable graft to PGA, and proliferated. After 4 weeks, the grafts were populated by both seeded BMCs and native ECs. Immunostaining showed that the graft not only contained ECs, but also contained SMCs at varying stages of development. With this evidence, the group concluded that this process ultimately leads to the generation of new blood vessels, with all of their components. After its success in dogs, this technology was applied to humans. Although the greatest need for TEVGs is in adults who require vascular or cardiac bypass or access for hemodialysis, another potential application of TEVG is its use in pediatric patients with congenital heart disease who require extra cardiac conduits. The problem with prosthetic conduits is that they do not grow with the © 2015 Wolters Kluwer Health, Inc. All rights reserved.
child and require reoperations for replacement. In addition to the previously discussed theoretical advantages of TEVGs, these grafts have the potential for growth with the patient which would obviate the need for reoperation. Shin’oka et al16 implanted BM-MNC-seeded PGA grafts into 42 human subjects between the ages of 1 and 24 for the treatment of congenital heart defects. After a mean follow up of 490 days, there was no evidence of thrombosis, stenosis, obstruction, or any other complications with the grafts. By the time of followup, no patients required the use of anticoagulation therapy. Because of the vascular nature and excellent hemodynamic performance of the grafts, the results of this study are very promising and suggest that further intervention with patient growth will not be necessary. This will be determined after long-term follow-ups. In 2010,17 after approximately 6 years, the 25 patients who were treated for single ventricle physiology with TEVG extracardiac total c avopulmonary connections had late-term follow-ups. Although there were no instances of aneurysm formation, calcification, rupture, or infection, 4 patients had graft stenosis and 1 developed a partial mural thrombosis. All of these complications were successfully treated without open surgery. The graft stenoses were treated with percutaneous angioplasty, and the mural thrombosis was treated with warfarin. Treatment of single-ventricle physiology with the TEVG approach of seeding BM-MNCs on a biodegradable scaffold, therefore, appears to be efficacious; however, further studies are necessary to determine the mechanism of graft stenosis, because this was the predominant cause of graft failure.
Neotissue Formation Hashi et al18 found that BM-MNCs resisted both a thrombotic response and intimal hyperplasia. This finding led many researchers to study the mechanism of neotissue formation leading to stenosis. In a murine model, Hashi et al18 and Hibino et al19 found that seeded BM-MNCs contribute to neovessel development through a paracrine mechanism causing ingrowth of surrounding cells. Using sexmismatched chimeric hosts, they demonstrated that the BM-MNCs themselves are not the source of neotissue, but that the adjacent vessels contribute ECs and SMCs, composing 93% of the neotissue. This suggests that neotissue formation is the result of the body’s natural repair mechanisms, not the differentiation of BM-MNCs as was previously thought.3,19,20 Naito et al21 analyzed biomechanical and biological properties of TEVGs implanted into mouse IVCs over a 24-week period. They found that within the first 4 weeks of the study, the majority of the biodegradable scaffold was replaced with an ECM made of collagen, and versican, which is important for cell migration. Over the course of 24 weeks, the graft further remodeled and developed to behave very similar to native IVC—stiffness of the www.cardiologyinreview.com | 237
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cardiology in Review • Volume 23, Number 5, September/October 2015
Jaspan and Hines
TEVG continue to decrease despite no change in thickness from 12 to 24 weeks. These mechanobiological properties need to be further studied to design second generation TEVGs with lower incidences of stenosis. Cleary et al3 describe how multiple variables are thought to contribute to cell growth and graft stenosis, including biomechanical properties of the scaffold, immune response, and signaling pathways involved in SMC proliferation. Currently, it is hypothesized that an inflammatory cascade initiated by cytokines and growth factors contributes to vascular remodeling. Controlling the host’s immune response is very important—there is a balance between excessive macrophage infiltration and complete macrophage inhibition, where the former can lead to occlusion, and the latter prevents proper neotissue development. Regulating the expression of growth factors and cytokines through microparticles, which mimic human BMCs embedded in the scaffold, seems to be a promising innovation for the future success of TEVGs. Controlled-release microparticles can potentially circumvent the need for seeding grafts. This work is currently underway.3
Scaffold Construction The biomechanical properties of the scaffold, which have significant effects on the success of the graft, are determined both by the type of polymer used and by the method of graft manufacture. New technologies allow for more precise regulation of fiber diameter, orientation, and density. These properties are very important for improving cell migration and attachment and can influence the development of stenosis.3 Sustaining adequate porosity is extremely important for vascular remodeling, in order for inflammatory cells to adhere to the graft and maintain paracrine signaling to produce an adequate ECM.22 Also important for maintaining an adequate ECM and SMC structure is the use of pulsatile bioreactors for the creation of a dynamic cell culture environment. One method of graft production, known as electrospinning, uses electrostatic interactions to spin nanofibers into grafts.23 Although very useful in producing nanoscale fibers from polymers, smaller fibers lead to decreased porosity, thus limiting ECM production. Bilayers, in which one layer is more porous and the other is less porous, are being tested for their success in avoiding this problem.22 de Valence et al24 tested a bilayered electrospun graft in 20 rat abdominal aortas. After 3 weeks, there was less than a 10% incidence of intimal hyperplasia, no aneurysms or thrombosis, and all 20 grafts were patent. These grafts have not yet been tested in humans. Maintaining dynamic load through the use of pulsatile bioreactors is important for the development of SMC and ECM structure in electrospun grafts. Thomas et al25 created an electrospun graft out of a gelatin and vinyl acetate copolymer seeded with rat vascular SMCs. They produced this graft both in static and in dynamic conditions for 7 days. To create a dynamic cell culture environment, a pulsatile bioreactor was set to mimic physiologic conditions. After 7 days, they found that compared with those grown in static conditions, TEVGs grown in pulsatile conditions secreted well-oriented SMCs and higher concentrations of collagen and elastin. The concentrations of elastin were found to be almost 80% of that of native tissue. Tissue grown under dynamic conditions was also stronger and had significantly higher burst pressures than those grown in static conditions. The authors suggest that these improvements in d ynamically grown grafts may be because of the increased opportunity for cells to adjust to pore diameter and grow into the scaffold. They conclude that pulsatile forces help modulate cell morphology and phenotypic plasticity of the grafts. Consistent with Thomas et al, Zhang et al26 demonstrate that TEVGs cultured under dynamic conditions are more successful than those cultured under static conditions. They used an electrospun silk-fibroin scaffold seeded with human SMCs and ECs and found that after 14 days, grafts grown under dynamic conditions had much more significant tissue formation, retention of 238 | www.cardiologyinreview.com
differentiated cell phenotype, cell alignment, and ECM production than those grown under static conditions. More work is necessary to better understand SMC phenotype and its control to further optimize electrospun TEVG development before they can be tested in humans. An alternative approach to electrospinning, which has not yet been used in clinical practice, is 3D bioprinting. This technology allows for the direct deposition of scaffold materials and cells to construct digitally designed 3D vessels. Much work needs to be done to achieve adequate compliance, tear strength, burst strength, and longterm stability before in vivo studies can begin. Based on the previous work, Hoch et al27 expect this to occur in the coming decade.
DECELLULARIZED MATRICES Decellularized matrices, derived from xenografts and homografts, can be used as scaffolds in the production of TEVGs. Decellularization is necessary to remove antigenic components.28 A decellularized vein matrix is a particularly good scaffold because it maintains a nonimmunogenic 3D ECM structure, which triggers differentiation among seeded cells.29 Kaushal et al30 demonstrated that decellularized porcine iliac vessels seeded with autologous ovine ECs could be successfully implanted into ovine carotid arteries and remain patent. Unseeded decellularized vessels occluded within 15 days, thus demonstrating the importance of seeding. Bertanha et al29 seeded decellularized vena cava with mesenchymal stem cells instead of ECs, because these could be rapidly obtained from adipose tissue. They demonstrated that, over the course of a 3-week period, in the presence of endothelial inductor growth factor and a 3D-scaffold, mesenchymal stem cells can deposit ECM and correctly differentiate into ECs. They even begin to organize into capillaries. This finding was supported by histology and general morphology analyses, and by the increased concentration of von Willebrand factor found in cells exposed to endothelial inductor growth factor, when compared with negative controls. In the future, these grafts can be strengthened with SMCs, which can then be experimented in vivo. Decellularized matrices are currently limited because of lack of control of architecture, and risk of viral transmission.3,31
CONCLUSION The development of TEVGs is very promising for the field of cardiovascular surgery. Although many factors, including our understanding of neotissue formation, must be improved before the technology can be fully implemented, much progress has been made in recent years. With the development of 3D-printed and electrospun TEVGs and recent studies better characterizing neotissue formation, new and improved grafts can be designed and tested in animal models. In the near-future, TEVGs will provide a viable alternative to traditional synthetic grafts and should increase long-term patency rates. REFERENCES 1. Chlupác J, Filová E, Bacáková L. Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery. Physiol Res. 2009;58(Suppl 2):S119–S139. 2. Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science. 1986;231:397–400. 3. Cleary MA, Geiger E, Grady C, et al. Vascular tissue engineering: the next generation. Trends Mol Med. 2012;18:394–404. 4. L’Heureux N, Pâquet S, Labbé R, et al. A completely biological tissue-engineered human blood vessel. FASEB J. 1998;12:47–56. 5. McAllister TN, Maruszewski M, Garrido SA, et al. Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study. Lancet. 2009;373:1440–1446. 6. Wystrychowski W, McAllister TN, Zagalski K, et al. First human use of an allogeneic tissue-engineered vascular graft for hemodialysis access. J Vasc Surg. 2014;60:1353–1357. 7. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery. 1978;84:498–504.
© 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cardiology in Review • Volume 23, Number 5, September/October 2015
8. Mazzucotelli JP, Moczar M, Zede L, et al. Human vascular endothelial cells on expanded PTFE precoated with an engineered protein adhesion factor. Int J Artif Organs. 1994;17:112–117. 9. Deutsch M, Meinhart J, Fischlein T, et al. Clinical autologous in vitro endothelialization of infrainguinal ePTFE grafts in 100 patients: a 9-year experience. Surgery. 1999;126:847–855. 10. Niklason LE, Gao J, Abbott WM, et al. Functional arteries grown in vitro. Science. 1999;284:489–493. 11. Mooney DJ, Mazzoni CL, Breuer C, et al. Stabilized polyglycolic acid fibrebased tubes for tissue engineering. Biomaterials. 1996;17:115–124. 12. Naito Y, Williams-Fritze M, Duncan DR, et al. Characterization of the natural history of extracellular matrix production in tissue-engineered vascular grafts during neovessel formation. Cells Tissues Organs. 2012;195:60–72. 13. Dahl SL, Kypson AP, Lawson JH, et al. Readily available tissue-engineered vascular grafts. Sci Transl Med. 2011;3:68–69. 14. Santos Fd, Andrade PZ, Abecasis MM, et al. Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods. 2011;17:1201–1210. 15. Matsumura G, Miyagawa-Tomita S, Shin’oka T, et al. First evidence that bone marrow cells contribute to the construction of tissue-engineered vascular autografts in vivo. Circulation. 2003;108:1729–1734. 16. Shin’oka T, Matsumura G, Hibino N, et al. Midterm clinical result of tissueengineered vascular autografts seeded with autologous bone marrow cells. J Thorac Cardiovasc Surg. 2005;129:1330–1338. 17. Hibino N, McGillicuddy E, Matsumura G, et al. Late-term results of tissue-engineered vascular grafts in humans. J Thorac Cardiovasc Surg. 2010;139:431–436. 18. Hashi CK, Zhu Y, Yang GY, et al. Antithrombogenic property of bone marrow mesenchymal stem cells in nanofibrous vascular grafts. Proc Natl Acad Sci U S A. 2007;104:11915–11920. 19. Hibino N, Villalona G, Pietris N, et al. Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel. FASEB J. 2011;25:2731–2739.
© 2015 Wolters Kluwer Health, Inc. All rights reserved.
The Current Status of TEVGs
20. Hibino N, Yi T, Duncan DR, et al. A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts. FASEB J. 2011;25:4253–4263. 21. Naito Y, Lee YU, Yi T, et al. Beyond burst pressure: initial evaluation of the natural history of the biaxial mechanical properties of tissue-engineered vascular grafts in the venous circulation using a murine model. Tissue Eng Part A. 2014;20:346–355. 22. Rocco KA, Maxfield MW, Best CA, et al. In vivo applications of electrospun tissue-engineered vascular grafts: a review. Tissue Eng Part B Rev. 2014;20:628–640. 23. Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials. 2008;29:1989–2006. 24. de Valence S, Tille JC, Giliberto JP, et al. Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration. Acta Biomater. 2012;8:3914–3920. 25. Thomas LV, Nair PD. The effect of pulsatile loading and scaffold structure for the generation of a medial equivalent tissue engineered vascular graft. Biores Open Access. 2013;2:227–239. 26. Zhang X, Wang X, Keshav V, et al. Dynamic culture conditions to generate silkbased tissue-engineered vascular grafts. Biomaterials. 2009;30:3213–3223. 27. Hoch E, Tovar GE, Borchers K. Bioprinting of artificial blood vessels: current approaches towards a demanding goal. Eur J Cardiothorac Surg. 2014;46:767–778. 28. Dahl SL, Koh J, Prabhakar V, et al. Decellularized native and engineered arterial scaffolds for transplantation. Cell Transplant. 2003;12:659–666. 29. Bertanha M, Moroz A, Almeida R, et al. Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factors. J Vasc Surg. 2014;59:1677–1685. 30. Kaushal S, Amiel GE, Guleserian KJ, et al. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001;7:1035–1040. 31. Ravi S, Chaikof EL. Biomaterials for vascular tissue engineering. Regen Med. 2010;5:107–120.
www.cardiologyinreview.com | 239
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
The Current Status of Tissue-Engineered Vascular Grafts Vita N. Jaspan and George L. Hines, MD
Abstract: Tissue-engineered vascular grafts (TEVGs) are currently being developed to overcome the limitations and complications associated with traditional synthetic grafts. This article aims to review the current status of research into the production and use of tissue-engineered grafts. TEVGs have a number of theoretical advantages over synthetic grafts. The results of animal and human studies have been promising, but more work must be done before TEVGs can replace traditional grafts. Key Words: tissue-engineered vascular grafts, sheet-based, scaffold-based, electrospinning (Cardiology in Review 2015;23: 236–239)
V
ascular grafts have been used in humans since the early 1900s to treat a variety of acquired and congenital cardiac and vascular conditions. Uses include creation of cardiac shunts in congenital heart defects, repair of aneurysms, repair of injured blood vessels, bypass of stenotic and occluded blood vessels, creation of hemodialysis access, and as creation of portosystemic shunts. The first known use of vascular grafts in humans was performed by Goyannes who used autologous popliteal vein graft to repair a popliteal aneurysm in 1906.1 Later, in 1948, Kunlin performed the first saphenous vein femoropopliteal bypass surgery.1 By the 1950s, synthetic materials came into use to compensate for the lack of available autologous vascular tissue. At this time, Voorhees, Blakemore, and DeBakey used Dacron grafts to treat aortic aneurysms.1 Smaller size Dacron and polytetrafluoroethylene (PTFE) grafts were then introduced in the 1960s and 1970s, and intravascular stents were first introduced in the 1970s and 1980s.1 Although very effective, prosthetic grafts and stents can result in complications including intimal hyperplasia, thrombosis, and infection, especially in arteries of small diameter. Theoretically, some of these problems could be avoided by using native vessels for grafting. Because autogenous tissue may not be available in sufficient size or quantity for use as vascular grafts, tissue-engineered vascular grafts (TEVGs) have been developed. TEVGs are grown using the patient’s own cells and are therefore, by definition, made from autologous tissue. Because they are native in origin, TEVGs should be able to more closely resemble a patient’s vasculature, including vasoreactivity and biomechanics, thus increasing the probability of long-term patency. TEVGs could also be very effective in pediatric patients, where they have the potential to grow and remodel with the patient. There are multiple approaches to the development of TEVGs, all of which are outgrowths of the first TEVG created by Weinberg and Bell,2 which consisted of bovine endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts, all within a tubular collagen matrix. This graft, unfortunately, was unable to withstand physiologic
From the Division of Vascular Surgery, Department of Thoracic and Cardiovascular Surgery, Winthrop University Hospital, Mineola, NY. The authors declare no conflict of interest in the preparation of this manuscript. Correspondence: George L. Hines, MD, Winthrop Cardiovascular Associates, 120 Mineola Blvd, Suite 300, Mineola NY 11501. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 1061-5377/15/2305-0236 DOI: 10.1097/CRD.0000000000000060
236 | www.cardiologyinreview.com
pressures. More recent approaches vary in the types of cells, types of scaffolds, and in the humoral and mechanical signals involved in the regulation and maintenance of the neotissue.3 The various approaches to the development of TEVGs are shown in Figure 1.
SHEET-BASED TISSUE ENGINEERING OF CELL SELF-ASSEMBLY VESSELS One method of TEVG generation, known as sheet-based engineering, has already been used clinically for the creation of hemodialysis access. In this method, sheets of fibroblasts are extracted from biopsies of the patient’s skin and superficial veins, wrapped around a stainless steel mandrel, and cultured for 10 weeks in a bovine serum. The mandrel is then removed, and the sheets are seeded with ECs to promote nonthrombogenicity.4 Production of these grafts takes between 6 and 9 months. Between 2004 and 2007, McAllister et al5 implanted sheet-based grafts in 10 different patients who had at least one previous access failure and who were considered to be at a high risk for graft failure. Of these, three-fourths were patent after 1 month, and three-fifths were patent after 6 months. Failures were secondary to dilatation and the development of aneurysms, despite the high burst strength and expected low immune response of the grafts. Although an encouraging result, these grafts are very costly, time consuming to produce and must be improved before they can replace traditional synthetic grafts. Wystrychowski et al6 recently implanted three sheet-based allogeneic grafts as brachial-artery arteriovenous shunts for hemodialysis access. This was the first use of allogeneic TEVGs in humans. The grafts were formed from cells extracted from 2 donors and were not endothelialized. After 7–12 weeks, all 3 grafts were punctured and used for hemodialysis access, at which time good blood flow was observed. No immunologic or inflammatory responses were observed. After approximately 4 months, intervention was required in all 3 grafts, primarily to correct stenoses of axillary and central veins. Within 7 months of implantation, all 3 grafts failed for a variety of reasons, including thrombosis, an unrelated infection, and physical injury. Other problems regarding slow-healing after puncture were noted, which the authors suggested were likely influenced by the lack of endothelialization of these grafts. This case study yielded very promising results regarding the potential for widespread TEVG use in humans. No infections or aneurysms developed in any of the grafts, but thrombosis and quick failure of all 3 grafts indicate that more work must be done to improve the durability of allogeneic TEVGs. This study also reveals that ECs may not be necessary to maintain mechanical stability of the graft, as was previously thought.
SCAFFOLD-BASED TECHNOLOGIES Most current strategies for the development of TEVGs involve scaffold-based technologies. In 1978, Herring et al7 proposed seeding ECs onto the surface of synthetic conduits. These grafts showed no advantage over traditional synthetic grafts. This approach was later refined when Mazzucotelli et al8 seeded ECs onto PTFE grafts with fibrinolytically inhibited fibrin glue, which improved patency of small-diameter vascular grafts. This improvement was because of the increased adhesion of cells to the graft. Using this strategy, Deutsch et al9 demonstrated a 9-year patency rate of 65% when compared
Cardiology in Review • Volume 23, Number 5, September/October 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cardiology in Review • Volume 23, Number 5, September/October 2015
The Current Status of TEVGs
FIGURE 1. Brief overview of the various approaches to tissue-engineered vascular graft (TEVG) development. with 16% for the control group who received untreated ePTFE grafts, for infrainguinal reconstructions. These synthetic materials cannot remodel, thus preventing the growth and vasoreactivity, which would increase their utility.3 For this reason, biodegradable synthetic polymer scaffolds are preferred, which degrade over time, leaving behind vessels that more closely resemble native tissue. Niklason et al10 cultured porcine aortic SMCs on a polyglycolic acid (PGA) fabric. PGA is a biodegradable material that begins to degrade after 2–4 weeks and is completely absorbed within 6 months of implantation. Poly-l-lactic acid can be copolymerized with PGA to regulate degradation rates.11 The investigators incubated the seeded scaffold in a pulsatile bioreactor to mimic physiologic conditions, which promoted the production of a robust extracellular matrix (ECM).10 Proper ECM development is vital to the success of the graft—improper development can result in stenosis and dilatation.12 The resulting vessel was vasoreactive and had burst pressures in excess of 2000 mm Hg.10 When Dahl et al13 transplanted a PGA graft seeded with human cells into baboons as arteriovenous conduits, an 88% patency was observed over a 6-month period. No aneurysmal dilatation or calcification was observed, and minimal intimal hyperplasia was evident. After 6 months, much remodeling occurred, and a loose, fibrous adventitia-like layer was visible. Despite promising results, the cell-culture technique is very time-consuming, limiting the utility of these technologies. Furthermore, the process of culturing cells requires the use of serum from nonhuman species, thus increasing the risk of contamination.14 To overcome these problems associated with in vitro cell cultures, Matsumura et al15 found that bone marrow derived mononuclear cells (BM-MNC) can develop into native-like vasculature. These cells are readily available in the iliac crest and can be isolated the day of surgery, thereby obviating the need for cell culture. First, they implanted these BM-MNC seeded grafts into the inferior vena cava (IVC) of dogs. Using immunohistochemistry, they confirmed that the seeded bone marrow cells (BMCs) adhered to the poly-l-lactic acid scaffold, an alternative biodegradable graft to PGA, and proliferated. After 4 weeks, the grafts were populated by both seeded BMCs and native ECs. Immunostaining showed that the graft not only contained ECs, but also contained SMCs at varying stages of development. With this evidence, the group concluded that this process ultimately leads to the generation of new blood vessels, with all of their components. After its success in dogs, this technology was applied to humans. Although the greatest need for TEVGs is in adults who require vascular or cardiac bypass or access for hemodialysis, another potential application of TEVG is its use in pediatric patients with congenital heart disease who require extra cardiac conduits. The problem with prosthetic conduits is that they do not grow with the © 2015 Wolters Kluwer Health, Inc. All rights reserved.
child and require reoperations for replacement. In addition to the previously discussed theoretical advantages of TEVGs, these grafts have the potential for growth with the patient which would obviate the need for reoperation. Shin’oka et al16 implanted BM-MNC-seeded PGA grafts into 42 human subjects between the ages of 1 and 24 for the treatment of congenital heart defects. After a mean follow up of 490 days, there was no evidence of thrombosis, stenosis, obstruction, or any other complications with the grafts. By the time of followup, no patients required the use of anticoagulation therapy. Because of the vascular nature and excellent hemodynamic performance of the grafts, the results of this study are very promising and suggest that further intervention with patient growth will not be necessary. This will be determined after long-term follow-ups. In 2010,17 after approximately 6 years, the 25 patients who were treated for single ventricle physiology with TEVG extracardiac total c avopulmonary connections had late-term follow-ups. Although there were no instances of aneurysm formation, calcification, rupture, or infection, 4 patients had graft stenosis and 1 developed a partial mural thrombosis. All of these complications were successfully treated without open surgery. The graft stenoses were treated with percutaneous angioplasty, and the mural thrombosis was treated with warfarin. Treatment of single-ventricle physiology with the TEVG approach of seeding BM-MNCs on a biodegradable scaffold, therefore, appears to be efficacious; however, further studies are necessary to determine the mechanism of graft stenosis, because this was the predominant cause of graft failure.
Neotissue Formation Hashi et al18 found that BM-MNCs resisted both a thrombotic response and intimal hyperplasia. This finding led many researchers to study the mechanism of neotissue formation leading to stenosis. In a murine model, Hashi et al18 and Hibino et al19 found that seeded BM-MNCs contribute to neovessel development through a paracrine mechanism causing ingrowth of surrounding cells. Using sexmismatched chimeric hosts, they demonstrated that the BM-MNCs themselves are not the source of neotissue, but that the adjacent vessels contribute ECs and SMCs, composing 93% of the neotissue. This suggests that neotissue formation is the result of the body’s natural repair mechanisms, not the differentiation of BM-MNCs as was previously thought.3,19,20 Naito et al21 analyzed biomechanical and biological properties of TEVGs implanted into mouse IVCs over a 24-week period. They found that within the first 4 weeks of the study, the majority of the biodegradable scaffold was replaced with an ECM made of collagen, and versican, which is important for cell migration. Over the course of 24 weeks, the graft further remodeled and developed to behave very similar to native IVC—stiffness of the www.cardiologyinreview.com | 237
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cardiology in Review • Volume 23, Number 5, September/October 2015
Jaspan and Hines
TEVG continue to decrease despite no change in thickness from 12 to 24 weeks. These mechanobiological properties need to be further studied to design second generation TEVGs with lower incidences of stenosis. Cleary et al3 describe how multiple variables are thought to contribute to cell growth and graft stenosis, including biomechanical properties of the scaffold, immune response, and signaling pathways involved in SMC proliferation. Currently, it is hypothesized that an inflammatory cascade initiated by cytokines and growth factors contributes to vascular remodeling. Controlling the host’s immune response is very important—there is a balance between excessive macrophage infiltration and complete macrophage inhibition, where the former can lead to occlusion, and the latter prevents proper neotissue development. Regulating the expression of growth factors and cytokines through microparticles, which mimic human BMCs embedded in the scaffold, seems to be a promising innovation for the future success of TEVGs. Controlled-release microparticles can potentially circumvent the need for seeding grafts. This work is currently underway.3
Scaffold Construction The biomechanical properties of the scaffold, which have significant effects on the success of the graft, are determined both by the type of polymer used and by the method of graft manufacture. New technologies allow for more precise regulation of fiber diameter, orientation, and density. These properties are very important for improving cell migration and attachment and can influence the development of stenosis.3 Sustaining adequate porosity is extremely important for vascular remodeling, in order for inflammatory cells to adhere to the graft and maintain paracrine signaling to produce an adequate ECM.22 Also important for maintaining an adequate ECM and SMC structure is the use of pulsatile bioreactors for the creation of a dynamic cell culture environment. One method of graft production, known as electrospinning, uses electrostatic interactions to spin nanofibers into grafts.23 Although very useful in producing nanoscale fibers from polymers, smaller fibers lead to decreased porosity, thus limiting ECM production. Bilayers, in which one layer is more porous and the other is less porous, are being tested for their success in avoiding this problem.22 de Valence et al24 tested a bilayered electrospun graft in 20 rat abdominal aortas. After 3 weeks, there was less than a 10% incidence of intimal hyperplasia, no aneurysms or thrombosis, and all 20 grafts were patent. These grafts have not yet been tested in humans. Maintaining dynamic load through the use of pulsatile bioreactors is important for the development of SMC and ECM structure in electrospun grafts. Thomas et al25 created an electrospun graft out of a gelatin and vinyl acetate copolymer seeded with rat vascular SMCs. They produced this graft both in static and in dynamic conditions for 7 days. To create a dynamic cell culture environment, a pulsatile bioreactor was set to mimic physiologic conditions. After 7 days, they found that compared with those grown in static conditions, TEVGs grown in pulsatile conditions secreted well-oriented SMCs and higher concentrations of collagen and elastin. The concentrations of elastin were found to be almost 80% of that of native tissue. Tissue grown under dynamic conditions was also stronger and had significantly higher burst pressures than those grown in static conditions. The authors suggest that these improvements in d ynamically grown grafts may be because of the increased opportunity for cells to adjust to pore diameter and grow into the scaffold. They conclude that pulsatile forces help modulate cell morphology and phenotypic plasticity of the grafts. Consistent with Thomas et al, Zhang et al26 demonstrate that TEVGs cultured under dynamic conditions are more successful than those cultured under static conditions. They used an electrospun silk-fibroin scaffold seeded with human SMCs and ECs and found that after 14 days, grafts grown under dynamic conditions had much more significant tissue formation, retention of 238 | www.cardiologyinreview.com
differentiated cell phenotype, cell alignment, and ECM production than those grown under static conditions. More work is necessary to better understand SMC phenotype and its control to further optimize electrospun TEVG development before they can be tested in humans. An alternative approach to electrospinning, which has not yet been used in clinical practice, is 3D bioprinting. This technology allows for the direct deposition of scaffold materials and cells to construct digitally designed 3D vessels. Much work needs to be done to achieve adequate compliance, tear strength, burst strength, and longterm stability before in vivo studies can begin. Based on the previous work, Hoch et al27 expect this to occur in the coming decade.
DECELLULARIZED MATRICES Decellularized matrices, derived from xenografts and homografts, can be used as scaffolds in the production of TEVGs. Decellularization is necessary to remove antigenic components.28 A decellularized vein matrix is a particularly good scaffold because it maintains a nonimmunogenic 3D ECM structure, which triggers differentiation among seeded cells.29 Kaushal et al30 demonstrated that decellularized porcine iliac vessels seeded with autologous ovine ECs could be successfully implanted into ovine carotid arteries and remain patent. Unseeded decellularized vessels occluded within 15 days, thus demonstrating the importance of seeding. Bertanha et al29 seeded decellularized vena cava with mesenchymal stem cells instead of ECs, because these could be rapidly obtained from adipose tissue. They demonstrated that, over the course of a 3-week period, in the presence of endothelial inductor growth factor and a 3D-scaffold, mesenchymal stem cells can deposit ECM and correctly differentiate into ECs. They even begin to organize into capillaries. This finding was supported by histology and general morphology analyses, and by the increased concentration of von Willebrand factor found in cells exposed to endothelial inductor growth factor, when compared with negative controls. In the future, these grafts can be strengthened with SMCs, which can then be experimented in vivo. Decellularized matrices are currently limited because of lack of control of architecture, and risk of viral transmission.3,31
CONCLUSION The development of TEVGs is very promising for the field of cardiovascular surgery. Although many factors, including our understanding of neotissue formation, must be improved before the technology can be fully implemented, much progress has been made in recent years. With the development of 3D-printed and electrospun TEVGs and recent studies better characterizing neotissue formation, new and improved grafts can be designed and tested in animal models. In the near-future, TEVGs will provide a viable alternative to traditional synthetic grafts and should increase long-term patency rates. REFERENCES 1. Chlupác J, Filová E, Bacáková L. Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery. Physiol Res. 2009;58(Suppl 2):S119–S139. 2. Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science. 1986;231:397–400. 3. Cleary MA, Geiger E, Grady C, et al. Vascular tissue engineering: the next generation. Trends Mol Med. 2012;18:394–404. 4. L’Heureux N, Pâquet S, Labbé R, et al. A completely biological tissue-engineered human blood vessel. FASEB J. 1998;12:47–56. 5. McAllister TN, Maruszewski M, Garrido SA, et al. Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study. Lancet. 2009;373:1440–1446. 6. Wystrychowski W, McAllister TN, Zagalski K, et al. First human use of an allogeneic tissue-engineered vascular graft for hemodialysis access. J Vasc Surg. 2014;60:1353–1357. 7. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery. 1978;84:498–504.
© 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cardiology in Review • Volume 23, Number 5, September/October 2015
8. Mazzucotelli JP, Moczar M, Zede L, et al. Human vascular endothelial cells on expanded PTFE precoated with an engineered protein adhesion factor. Int J Artif Organs. 1994;17:112–117. 9. Deutsch M, Meinhart J, Fischlein T, et al. Clinical autologous in vitro endothelialization of infrainguinal ePTFE grafts in 100 patients: a 9-year experience. Surgery. 1999;126:847–855. 10. Niklason LE, Gao J, Abbott WM, et al. Functional arteries grown in vitro. Science. 1999;284:489–493. 11. Mooney DJ, Mazzoni CL, Breuer C, et al. Stabilized polyglycolic acid fibrebased tubes for tissue engineering. Biomaterials. 1996;17:115–124. 12. Naito Y, Williams-Fritze M, Duncan DR, et al. Characterization of the natural history of extracellular matrix production in tissue-engineered vascular grafts during neovessel formation. Cells Tissues Organs. 2012;195:60–72. 13. Dahl SL, Kypson AP, Lawson JH, et al. Readily available tissue-engineered vascular grafts. Sci Transl Med. 2011;3:68–69. 14. Santos Fd, Andrade PZ, Abecasis MM, et al. Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods. 2011;17:1201–1210. 15. Matsumura G, Miyagawa-Tomita S, Shin’oka T, et al. First evidence that bone marrow cells contribute to the construction of tissue-engineered vascular autografts in vivo. Circulation. 2003;108:1729–1734. 16. Shin’oka T, Matsumura G, Hibino N, et al. Midterm clinical result of tissueengineered vascular autografts seeded with autologous bone marrow cells. J Thorac Cardiovasc Surg. 2005;129:1330–1338. 17. Hibino N, McGillicuddy E, Matsumura G, et al. Late-term results of tissue-engineered vascular grafts in humans. J Thorac Cardiovasc Surg. 2010;139:431–436. 18. Hashi CK, Zhu Y, Yang GY, et al. Antithrombogenic property of bone marrow mesenchymal stem cells in nanofibrous vascular grafts. Proc Natl Acad Sci U S A. 2007;104:11915–11920. 19. Hibino N, Villalona G, Pietris N, et al. Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel. FASEB J. 2011;25:2731–2739.
© 2015 Wolters Kluwer Health, Inc. All rights reserved.
The Current Status of TEVGs
20. Hibino N, Yi T, Duncan DR, et al. A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts. FASEB J. 2011;25:4253–4263. 21. Naito Y, Lee YU, Yi T, et al. Beyond burst pressure: initial evaluation of the natural history of the biaxial mechanical properties of tissue-engineered vascular grafts in the venous circulation using a murine model. Tissue Eng Part A. 2014;20:346–355. 22. Rocco KA, Maxfield MW, Best CA, et al. In vivo applications of electrospun tissue-engineered vascular grafts: a review. Tissue Eng Part B Rev. 2014;20:628–640. 23. Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials. 2008;29:1989–2006. 24. de Valence S, Tille JC, Giliberto JP, et al. Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration. Acta Biomater. 2012;8:3914–3920. 25. Thomas LV, Nair PD. The effect of pulsatile loading and scaffold structure for the generation of a medial equivalent tissue engineered vascular graft. Biores Open Access. 2013;2:227–239. 26. Zhang X, Wang X, Keshav V, et al. Dynamic culture conditions to generate silkbased tissue-engineered vascular grafts. Biomaterials. 2009;30:3213–3223. 27. Hoch E, Tovar GE, Borchers K. Bioprinting of artificial blood vessels: current approaches towards a demanding goal. Eur J Cardiothorac Surg. 2014;46:767–778. 28. Dahl SL, Koh J, Prabhakar V, et al. Decellularized native and engineered arterial scaffolds for transplantation. Cell Transplant. 2003;12:659–666. 29. Bertanha M, Moroz A, Almeida R, et al. Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factors. J Vasc Surg. 2014;59:1677–1685. 30. Kaushal S, Amiel GE, Guleserian KJ, et al. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001;7:1035–1040. 31. Ravi S, Chaikof EL. Biomaterials for vascular tissue engineering. Regen Med. 2010;5:107–120.
www.cardiologyinreview.com | 239
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
