- Email: [email protected]
The development and translation of the tissue-engineered vascular graft
Journal of Pediatric Surgery (2011) 46, 8–17
www.elsevier.com/locate/jpedsurg
Jay and Margie Grosfeld Lecture
The development and translation of the tissue-engineered vascular graft Christopher K. Breuer Yale University, New Haven, CT 06520, USA Received 27 August 2010; accepted 30 September 2010
Key words: Tissue-engineered vascular grafts; Development; Translation
Abstract This lecture will define the classic tissue engineering paradigm, describe cell trafficking with regard to neotissue formation, and explain the role of the host in neotissue formation. © 2011 Elsevier Inc. All rights reserved.
Dr Georgeson, thank you for the introduction, and congratulations on your APSA presidency. It is well deserved. You are a great leader in our field, a pioneer, and definitely not a cowboy or desperado.
Funding sources: American Pediatric Surgical Association Foundation Enrichment Grant American Surgical Association Foundation Fellowship Research Award NIH K08-HL083980 NIH R01-HL098228. E-mail address: [email protected]. 0022-3468/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.09.058
Dr and Mrs Grosfeld, I wanted to thank you for your continued support of APSA. Dr Grosfeld, you are the consummate pediatric surgeon and a role model for us all. I owe you a personal debt of gratitude for your support of my research through your work in the American Surgical Association and American Pediatric Surgical Association foundations. Continued support by philanthropic organizations such as the APSA foundation is critical to the continued development of our next generation of surgeon-scientists. In 1993, I was a surgical research fellow in Jay Vacanti's tissue engineering laboratory (Fig. 1). He and Dr Bob Langer were pioneers in a field that would come to be known as regenerative medicine. They had come up with the notion of creating tissue from its cellular components by seeding cells onto a biodegradable scaffold. The resulting neotissue could be used for a variety of reconstructive surgical applications. It was a magical time in the development of the field of tissue engineering, which seemed capable of blurring the boundaries between science and science fiction. The human ear on the back of the mouse (or the auriculosauris as it came to be known) became the icon for this burgeoning science. The auriculosauris project was a seminal work and proof-of-principle experiment that highlighted the immense potential of this field.
Tissue-engineered vascular grafts
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Fig. 1
Vacanti Laboratory circa 1995.
While I had absolutely nothing to do with the design or performance of this elegant experiment, I was the first to recognize that these animals were deaf and could not hear a thing with their tissue-engineered ears. However, since I am an innovator, I recognized there were other uses for this model. The potential was enormous. I am humbled when I think of how far the field of earring design has come due to the development of this technology (Fig. 2). On a more serious note, why do research? To be completely honest with you, I went into the lab for all of the wrong reasons, as a means to an end. I wanted to be a pediatric surgeon and it had been explained to me in no uncertain terms that performing
Fig. 2
Auriculosauris with earring.
a productive research fellowship was an unwritten prerequisite, a hurdle if you will. Fortunately for me, before working in the lab, I took some time off from my general surgical residency to do some additional surgical training in order to be certain of my career choice. I worked as a junior resident on the pediatric surgical service at the Children's Hospital in Boston. Mike Caty was the chief resident and Steve Fishman the junior fellow. During this time I discovered that I loved pediatric surgery. I enjoyed working with children and their families and I especially liked operating on sick kids and making them better. However, I was equally frustrated by our inability to care for a number of children with a variety of serious surgical conditions for which there were no cures. I was particularly touched by a child who developed short bowel syndrome after undergoing an extensive bowel resection for necrotizing enterocolitis. I watched in horror as this child died an agonizingly slow death and was equally distraught to watch the devastating effect it had on her family. This experience led me to the conclusion that for me, it wasn't enough to take care of my patients. I needed to do something to advance the field and help improve our ability to treat diseases for which there were no cures. Ultimately this led me to consider work in translational research. I started in the lab with an abundance of enthusiasm and essentially no skill. For my project I had decided to follow in the footsteps of Greg Organ, the research fellow who preceded me in the Vacanti lab, and a future pediatric surgeon. I wanted to try to build upon his work tissueengineering intestine. Greg had performed some of the
10 foundational work in the field. He developed methods for isolating and culturing enterocytes, in addition to seeding and implanting tissue-engineered constructs in vivo. It was a perfect solution for my desire to advance the field through translational research. Unfortunately, after completing my first year in the laboratory, I had absolutely nothing to show for my work. I had not generated a single piece of usable data. One possible explanation was that Greg was simply wrong and that his idea would never work. A more likely explanation was simply that I wasn't a very good scientist. A partial listing of the 107 articles that both build upon or substantially advance Greg's work give credence to the notion that I wasn't a very good researcher. And so I learned the first and most important lesson of research: embrace failure. Whenever a candidate inquires about working in my lab, I always explain to them that I will teach them to fail spectacularly. It is through failure that we learn our greatest lessons and make our best discoveries. Failure, or perhaps more precisely fear of failure, fuels creativity, a scientist's best and most important attribute. After my first year in the lab, I went back to the drawing board. I made 2 lists; the first described what we could not do in the field of tissue engineering in 1994. We couldn't create capillary networks de novo, we couldn't innervate neotissue, and we couldn't grow many of the different cell types needed to make many of the organs we were interested in making. I thought I might consider working on a different project, but what to do? Upon further reflection I started to think about what we could do so I made a second list. We were really good at making ears, or other structural tissues made of bone or cartilage. We had fairly sophisticated methods for fabricating scaffolds. And while we couldn't grow a number of different cell types, even I could grow fibroblasts (the dandelion of the cell world), smooth muscle cells weren't that hard either, and since our lab was in close proximity to Judah Folkman's lab, endothelial cells practically walked up the stairs and into our incubators. If I couldn't grow intestine perhaps there was a more appropriate project.
1. Tissue engineering part II: cardiovascular tissue engineering After some thought, I concluded that I could try making tissue-engineered heart valves and blood vessels. Surprisingly this time my research worked. Here is a photo of the first tissueengineered heart valve that I made and implanted in a lamb animal model (Fig. 3). And so I learned the second lesson of research: be open to serendipity. Or as my aunt once told me, “sometimes when God closes doors He opens windows.” The development of a man-made vascular graft with growth potential would have important implications for neonates and children. Within the field of pediatric surgery, there are several important situations in which the ability to tissue engineer a blood vessel would have great utility. However, from a public health perspective this is not a major problem. Congenital heart
C.K. Breuer
Fig. 3
Tissue-engineered heart valve leaflet.
disease on the other hand is an enormous public health issue affecting nearly 1% of all live births. Despite significant advances in the treatment of congenital heart disease, it remains a leading cause of death in the newborn period. I became particularly interested in a group of diverse structural anomalies that result in a severe form of congenital heart disease that results in single-ventricle physiology. Without surgical intervention, single-ventricle cardiac anomalies are associated with 70% mortality in the first year of life while survival to adulthood is rare. Fortunately, due to significant advances in the field of congenital heart surgery, patients born with single-ventricle cardiac anomalies have markedly improved longevity and improved quality of life. In order to fix patients born with single-ventricle anomalies the surgeon must perform a series of staged operations in which the plumbing is rearranged in such a way so that the single functional ventricle pumps oxygenated blood to the body where the oxygen is delivered and then the deoxygenated blood is returned directly to the pulmonary artery where it passively passes through the pulmonary circulation and is reoxygenated. This operation is referred to as a Fontan operation. Unfortunately, like most congenital heart operations, it requires the need for synthetic grafts or patches in order to complete the operation. Complications arising from the use of synthetic grafts are a leading cause of postoperative morbidity and mortality. Synthetic grafts are a significant source of thromboembolic complications, they have poor durability due to neointimal hyperplasia and ectopic calcification, they are susceptible to infections, and they lack growth potential, which is particularly problematic in children. Occasionally, a child is born with a single-ventricle anomaly that is amenable to primary repair without the use of a synthetic conduit. This small subset of patients does quite well and has been the impetus for our work. My research focuses on the creation of tissue-engineered vascular grafts that can be created by seeding an individual's own cells onto a biodegradable scaffold. The scaffold provides sites for cell attachment and Three-dimensional space for cell growth. As the neotissue forms the scaffolding degrades ultimately creating a neovessel without any synthetic components.
Tissue-engineered vascular grafts Our scaffold is fabricated from polyglycolic acid fibers, which are woven into a tube, and coated with a 50:50 copolymer of polylactic acid and polycaprolactone. It degrades by hydrolysis losing its biomechanical integrity in 8 weeks. Total fiber degradation takes approximately 6 months. Our cell source is autologous bone marrow–derived mononuclear cells obtained by performing a bone marrow aspirate and separating the bone marrow–derived mononuclear cell fraction using density centrifugation in Ficoll. It might seem strange that we make blood vessels from bone marrow. Originally we didn't. We previously made tissue-engineered vascular grafts by performing a biopsy of an individual's own blood vessel and then isolated the endothelial cells, smooth muscle cells, and fibroblasts which we then expanded in culture. When we had grown enough cells, we seeded our scaffolds and let the seeded scaffold grow in an incubator to allow time for cell attachment. Using this technique we could make a tissue-engineered vascular graft in 2 to 3 months. This long period of time limited the clinical utility of this technique. Additionally, prolonged periods of cell culture increased the chance of contamination or even malignant dedifferentiation of the cells. We also discovered that sick people have sick cells that sometimes couldn't be grown in culture. So we began to explore alternative cell sources and came upon bone marrow– derived mononuclear cells. These cells were available in great abundance so as to preclude the need for cell culture, dramatically reducing the time it took us to create a tissueengineered vascular graft from several months to several hours. We seed a concentrated cell suspension onto the scaffold and incubate the seeded scaffold in autologous serum (also obtained from the bone marrow aspirate) for 2 hours in order to allow for cell attachment. We have studied our tissue-engineered vascular graft in a variety of animal models. In order to evaluate the growth potential of the tissue-engineered vascular graft, we inserted it as an intrathoracic IVC interposition graft in a juvenile lamb model. The IVC replacement model is a validated animal model for investigating the use of grafts in the high-flow, lowpressure circulatory systems such as the Fontan circulation. The lamb model is the Food and Drug Administration's preferred model for investigating biological vascular grafts for use in congenital heart surgery due to the lamb's propensity for accelerated ectopic calcification, which is the leading cause of failure for biological conduits used in the repair of congenital cardiac anomalies. Next we serially monitored the tissueengineered vascular grafts using MRI. Using computer generated 3-dimensional imaging, we were able to superimpose images of the tissue-engineered vascular grafts at varying time points and thereby showed that the tissue-engineered vascular grafts increased in size over time. The increase in size was symmetrical and proportional to the increase in size of the surrounding vasculature suggesting growth instead of aneurysmal dilation [1]. In 2003, I came to Yale University as part of its tissue engineering initiative. Yale is a fairly small institution and compensates for its size by recruiting faculty in areas of
11 thematic research so as to optimize and build upon potential synergies. I have been repeatedly instructed by my new mentors to focus. I was told if I truly wanted to advance the field I would need to dig deeper and become more specialized. I needed to develop expertise in vascular biology, biomedical engineering, in addition to surgery. And so I learned the third lesson of research: Focus, focus, focus. The corollary to “focus, focus, focus” is that multidisciplinary collaborations are critical for success particularly for complex translational research projects. This requires certain leadership skills for it is akin to herding cats. We began to further characterize the structure of our tissue-engineered vascular grafts. We used histological stains and immunohistochemical techniques to demonstrate that the neovessels that resulted from the tissue-engineered vascular grafts were comprised of a monolayer of endothelial cells lining the luminal surface of the vessel surrounded by concentric layers of smooth muscle cells embedded in an extracellular matrix rich in both collagen and elastin similar to the native IVC. Neovessels even express Eph B4 the molecular determinant of venous development. During embryology a blood vessel is not considered a vein until Eph B4 is expressed. These findings suggest that neovessels are truly veins and not simply viable conduits. In 2001, Toshi Shinoka, my lab partner and the individual with whom I developed the basic techniques for tissueengineering blood vessel, reported the first clinical application of vascular tissue engineering [2]. He used a patch to perform a pulmonary artery angioplasty in a child undergoing an operation for pulmonary artery stenosis. He subsequently
Fig. 4 Three-dimensional CT of a tissue-engineered vascular graft 3 years after implantation. Reprinted from J Thorac Cardiovasc Surg 139(2) Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. 431-6 (2010).
12
C.K. Breuer
performed the first pilot study investigating the use of tissueengineered vascular grafts in congenital heart surgery. Here we see an image of a tissue-engineered vascular graft 3 years after implantation demonstrating a widely patent conduit (Fig. 4). In 2007, I was fortunate to be reunited with Dr Shinoka at Yale where he was recruited to be the head of the congenital heart surgery program. This highlights the fourth lesson of research: surround yourself with good people. Good not only in the sense of capable, but more importantly, good in the sense of a person with similar values, a strong work ethic, and complementary goals. This also applies to the selection of people who work for you. I have been fortunate to select research fellows who are uniformly excellent and are a real source of pride for me and make coming to work a pleasure. For a physician scientist it is also critical to surround yourself with great partners who understand and appreciate the role of research in medicine. With Dr Shinoka on board, we decided to compile all of our preclinical and clinical data in order to submit an application to the FDA for the first clinical trial in the United States evaluating the use of tissue-engineered vascular grafts in humans. This table summarizes the early (1-year) follow-up on 25 patients who had undergone extracardiac total cavopulmonary connection (EC TCPC) modified Fontan surgery using
Table 1
a tissue-engineered vascular graft (Table 1). There were no graft-related deaths and all tissue-engineered vascular grafts were patent and intact. The only complication was a partial mural thrombus that was successfully treated with coumadin. This table summarized the late (5- to 7-year follow-up) on this cohort of patients (Table 2). Again there was no graftrelated mortality. All the tissue-engineered vascular grafts were patent and intact. There were no new thromboembolic complications; however, 4 patients developed stenosis requiring angioplasty. This raises the obvious question: What is the rate of stenosis for PTFE grafts (the most commonly used grafts in Fontan surgery)? The incidence of stenosis in this cohort is poorly studied and difficult to ascertain from the literature. As in our series, EC TCPC conduit stenosis is typically asymptomatic and will not be detected unless the patient is imaged, which is not routinely done in this patient population. This results in underreporting. All patients in our study were routinely imaged as part of the investigative protocol. There are only 2 papers in the literature that serially monitor EC TCPC conduits using the same imaging modality [4,5]. The first is a large series from Italy, which selectively and randomly imaged a fraction of their cohort using serial MRI. They noted an 18% to 32% narrowing of the conduit within the
Patient status 1 year after TEVG implantation
Patient
Age at operation (y)
Graft type
Graft size (cm)
Patient status
Graft status
Graft patency
Graft-related complications
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
2 1 7 21 4 12 17 19 3 2 2 13 2 2 2 2 24 1 11 2 3 4 4 13 2
PLA PLA PLA PLA PLA PLA PLA PLA PLA PLA PGA PLA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA
16 20 18 24 20 24 24 22 12 16 16 20 16 18 12 16 18 16 18 16 16 18 18 16 18
Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive
Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact
Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent
None None None None None None None None None None None None Thrombosis None None None None None None None None None None None None
Early results of clinical trial [3]. TEVG indicates tissue-engineered vascular graft; PLA, poly-L-lactic acid: PGA, polyglycolic acid. Reprinted from J Thorac Cardiovasc Surg 139(2) Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. 431-6 (2010).
Tissue-engineered vascular grafts Table 2
13
Patient late-term status after TEVG implantation
Patient
Age of operation
Graft type
Graft size (cm)
Patient status
Graft status
Graft patency
Graft-related complications
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
2 1 7 21 4 12 17 19 3 2 2 13 2 2 2 2 24 1 11 2 3 4 4 13 2
PLA PLA PLA PLA PLA PLA PLA PLA PLA PLA PLA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA
16 20 18 24 20 24 24 22 12 16 20 16 16 18 12 16 18 16 18 16 16 18 18 16 18
Alive Alive Alive Alive Alive Alive Alive Dead Alive Dead Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead
Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact
Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent
None None Stenosis a None None None None None Stenosis a None None Stenosis a Thrombosis None None None None Stenosis a None None None None None None None
Late results of clinical trial [3]. TEVG indicates tissue-engineered vascular graft; PLA, poly-L-lactic acid; PGA, polyglycolic acid. Reprinted from J Thorac Cardiovasc Surg 139(2) Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. 431-6 (2010). a This patient underwent successful angioplasty.
first 6 months after implantation, which tended to stabilize and not to progress in most conduits after this time point [4]. The second paper was from a large Korean cohort, which was serially monitored using serial angiography. This group noted similar findings demonstrating around a 20% narrowing after the first 6 months after implantation [5]. Furthermore, the optimal treatment for asymptomatic stenosis has yet to be determined. In some studies, stenotic EC TCPC conduits were selectively treated with angioplasty; however, most EC TCPC conduit stenosis is treated with surgical graft replacement, which is associated with a 3% to 5% mortality. All 4 of our patients who developed stenosis were successfully treated with angioplasty or angioplasty and stenting. Overall these results compare favorably with synthetic vascular grafts. Synthetic vascular grafts appear to have a higher incidence of thromboembolic complications, while tissue-engineered vascular grafts have a higher incidence of stenosis. Tissue-engineered vascular grafts, however, are the only man-made grafts with growth potential. These clinical data have proven useful in directing our research efforts. In an attempt to rationally design an improved second-generation tissue-engineered vascular graft, we are currently investigating the mechanisms underlying tissue-engineered vascular graft stenosis.
Data obtained using large animal models provided us with some insights into the process of neovessel formation. Evaluation of histological specimen obtained over a 6-month time course demonstrated that neotissue forms both in and on the scaffold. Over time the scaffold degrades and the layers fuse and mature creating a laminated structure possessing a neointima, neomedia, and neoadeventitia. Understanding what factors control this process and which factors are important in the formation of stenosis is critical to our work. Over the past 5 years my laboratory has devoted significant effort toward developing a mouse model to study neovessel formation and tissue-engineered vascular graft stenosis. It may seem counterintuitive to develop a mouse model after having already translated our work from the bench to the clinic; however, there are a large number of molecular reagents that are readily available only for mouse work. These reagents are critical to this sort of work. Additionally, the use of a murine model has the added benefit of being able to perform experiments more efficiently and more cost effectively. To put this in perspective, in our first 10 years of research, we performed less than 200 implants; we currently perform over 500 per year. This allows us to ask and answer questions very rapidly. As a first step in developing a mouse model, we needed to create a method for fabricating the scaffold on a much smaller
14 scale. Jason Roh, a Yale medical student, developed a method for making the tissue-engineering scaffolds using the same design and materials as in the clinical trial but on a much smaller scale. The resulting scaffolds have the same porosity and degradation properties as the scaffolds used in the clinical trial but with diameters between 500 and 1000 μm, which enable implantation into the mouse vasculature. Using microsurgical technique, the tissue-engineered vascular graft can be implanted as an IVC interposition graft. We can then use micro-CT angiography to monitor neovessel formation. We have demonstrated that the tissue-engineered vascular grafts undergo the same remodeling over time that we have seen in large animal models and that the resulting neovessel resembles the native IVC both histologically and grossly. Once again, using immunohistochemical staining techniques, we have demonstrated that the neovessel is composed of a monolayer of endothelial cells lining the luminal surface surrounded by concentric layers of smooth muscle cells embedded in an exrtracellular matrix similar to that of the native IVC [6]. We have used time-course studies to break this process into phases. In the first phase, mononuclear cells invade the scaffold-forming macrophages. The concentration of macrophages in the scaffold initially increases. Endothelial cells and smooth muscle cells begin to form a neointima and neomedia on the inner surface of the scaffold. The scaffold itself gives rise to the neoadventitia. As the scaffolding degrades the vascular neotissue remodels, ultimately forming a neovessel that resembles the native IVC [7]. The first question we attempted to answer using the murine model is what is the role of cell seeding? Using microCT angiography we were able to demonstrate that seeded TEVG remained widely patent while unseeded TEVG tended to fail. The TEVG occlusion developed gradually as demonstrated by the formation of collateral blood vessels. Histological evaluation of the TEVG demonstrated that the mechanism of graft failure is stenosis. Thus, cell seeding improves patency by decreasing the incidence of stenosis. The next question we asked is what is the identity of the seeded cells? Bone marrow–derived mononuclear cells are composed of a heterogeneous population of mononuclear cells. We used multicolor florescent activated cell sorting to identify various subpopulations of cells within the bone marrow–derived mononuclear cell population. We had always assumed that the bone marrow–derived mononuclear cells were a rich source of vascular stem cells such as endothelial progenitor cells or mesenchymal stem cells; however, when we quantified the actual number of stem cells in the bone marrow–derived mononuclear cell population, it became obvious to us that thus was not the case. When we calculated the number of stem cells seeded onto the scaffold, we noted that only a few of these stem cells were attached to any single tissue-engineering scaffold [7]. What is the fate of the seeded cells? In order to determine the fate of the seeded cells, we created a chimera by seeding human bone marrow–derived mononuclear cells onto the tissue-engineering scaffold and implanted the human TEVG
C.K. Breuer into an immunosuppressed SCID-beige mouse. SCID-beige mice lack an adaptive immune response and readily tolerate xenotransplantation. Next we harvested the TEVG over a time course and tracked the seeded cells using human-specific immunohistochemical stains. We demonstrated that the human cells disappeared within 3 to 7 days after implantation. These findings flew in the face of the classic tissue-engineering paradigm in which the seeded cells are viewed as the building blocks of neotissue and were quite a surprise. We validated these findings using human-specific molecular probes, again showing that the seeded cells disappear. What are the cellular components of vascular neotissue? Since the seeded cells were not the building blocks of the vascular neotissue, what were the cells? Immunohistochemical analysis demonstrated that during the early phase of neotissue formation the vast majority of cells were host-derived macrophages. Quantitative analysis of the macrophage infiltrate demonstrated that cell seeding affected the degree of macrophage infiltration and that the degree of macrophage infiltration also correlated with the degree of stenosis suggesting that the host-derived macrophages may play a role in neovessel formation and the development of tissue-engineered vascular graft stenosis. Thus, we concluded that neovessel formation and the development of tissue-engineered vascular graft stenosis are inflammation-mediated processes. What are the molecular mechanisms of neovessel formation? How do the seeded cells exert such a powerful biological effect if they disappear within a few days after implantation? One possible explanation is that they exert a paracrine effect by initiating a cascade of events that ultimately lead to neovessel formation. In order to identify candidate cytokines important in this process, we measured cytokine production by the seeded tissue-engineered vascular grafts in vitro. We noted that the seeded cells produced relatively large quantities of MCP-1 (monocyte chemoattractive protein-1), a powerful chemokine that attracts macrophages wherever it is released. We hypothesized that this may play an important role in neovessel formation and the development of tissue-engineered vascular graft stenosis. In order to determine if MCP-1 production by the seeded cells was critical to neovessel formation and the development of tissue-engineered vascular graft stenosis, we took advantage of the fact that we had developed a mouse model and could therefore use transgenics. Transgenic mice are genetically engineered and bred to have specific genetic abnormalities. Knockout mice lack a specific gene. In the case of a MCP-1 knockout or null mouse, the MCP-1 gene is missing from every cell in the animal's body. Wild type refers to control mice, which are syngeneic with the exception of the knockout gene, which is present. We isolated bone marrow–derived mononuclear cells from either MCP-1 knockout mice or wild-type mice and used them to make TEVG, which were implanted into SCID-beige mice and harvested over a time course. Morphometric evaluation of the grafts demonstrated that the TEVG created using the MCP-1 knockout cells had narrower lumens and behaved
Tissue-engineered vascular grafts more like unseeded TEVG and less like seeded TEVG, suggesting MCP-1 was necessary for the process of neotissue formation and the development of TEVG stenosis. In order to see if MCP-1 was sufficient for the process of neovessel formation and the development of TEVG stenosis, we used controlled release technology to crate MCP-1 eluting microspheres. A microsphere is a microscopic particle created from a biodegradable material. A protein (in this case MCP-1) could be encapsuled in the microsphere. When encapsulated, the protein is in a state of suspended animation, but when released, it can exert its biologic effect and be degraded. We then incorporated the MCP-1 eluting microspheres into the design of our tissue-engineering scaffold creating an MCP-1 eluting tissue-engineered vascular graft. The MCP-1 eluting tissueengineering scaffold eluted MCP-1 at a rate and duration similar to the amount and duration of MCP-1 production by the seeded cells. Morphometric evaluation of the biomimetic TEVG demonstrated that the MCP-1 eluting scaffolds had larger diameters similar to the seeded TEVG and less like the unseeded TEVG. These findings suggested that MCP-1 is a critical player in neovessel formation. We currently are investigating the roles of other cytokines including PDGF and VEGF in this process in an attempt to better understand the mechanisms of neovessel formation and the development of TEVG stenosis. In our current model of neovessel formation, we believe that the seeded bone marrow–derived mononuclear cells release MCP-1 that attracts monocytes from the circulation into the scaffold where they become macrophages. The macrophages, in turn, release PDGF and VEGF which induce in-growth of the neighboring smooth muscle cells and endothelial cells. As the scaffold degrades, the macrophages begin to disappear and the vascular neotissue forms continuously remodeling until the scaffolding is completely gone and the neovessel is formed. Thus, at least in our model system, we have disproven the notion that the neovessel is formed from the seeded cells as originally proposed
Fig. 5
15 according to the classic tissue engineering paradigm in which the seeded cells are viewed as the building blocks of neotissue. Instead, these findings suggest that the mechanism of neovessel formation is regeneration of the blood vessel sort of like a salamander regrows its tail. This sort of mechanistic understanding has important implications for the rationale design of improved second generation TEVG. We recently submitted an application to the FDA supporting the performance of the first clinical trial in the United States investigating the use of TEVG in humans. We hope to bridge the chasm between the research and clinical practice and hopefully improve our ability to care for our patients. When I first began this project I assumed that there was a streamlined process with instructions that enabled a seamless transition from the bench to the clinic. Nothing could have been farther from the truth. This diagram outlines the path typically taken in order to bring a product from the bench to the clinic (Fig. 5). What had never been explained to me was that this is all considered proprietary information, which companies typically will not share. To make matters worse, there were no FDA-approved TEVG, so there was no president upon which to build. We needed to blaze our own trail following an extensive but vague roadmap. Four years later I am please to announce that we recently received approval from the FDA for performing our study. Obtaining approval was a long and arduous process; a single copy of our application was over 3000 pages. And so I learned the fifth lesson of research: work hard. There is no substitute. As I prepare to begin our clinical investigation, I realize that my research is truly only just beginning. I think of 2 papers that have dramatically altered my career and directed my actions. The first is Toshi Shinoka's initial case report in the New England Journal of Medicine describing the first clinical use of a TEVG [2]. It's hard to describe what a wonderful feeling it is to see an idea come to clinical fruition
Regulatory pathway for medical product development and translation (adapted from FDA Web site).
16 and be used to help a patient. The second article was published in a less prestigious journal and describes the research of one of my contemporaries who designed and developed a tissue-engineered heart valve created by seeding human cells onto a decellularized porcine heart valve, essentially using the extracellular matrix as a tissueengineering scaffold [8]. The investigators had performed some very exciting preclinical work demonstrating the feasibility of this technology and went on to perform a pilot study in humans. Four of the initial 6 patients implanted with the tissue-engineered heart valve died. The remaining 2 patients had to have their tissue-engineered heart valves removed and replaced with artificial heart valves. Post hoc analysis demonstrated that the pig heart valve had not been completely decellularized. The investigators had implanted a xenograft heart valve, which had been promptly rejected. This article highlights the critical role of proper regulatory control in any study. I am reminded that the road to perdition is paved with good intentions. We have all sworn a Hippocratic oath to first do no harm. A literal interpretation of this statement would bring the practice of surgery to a screeching halt. In fact, in the original Hippocratic oath, surgery was strictly forbidden. A closer reading of the original text suggests that this is not the idea that Hippocrates was originally trying to convey. Instead he was trying to insist that it is a physician's duty to try to achieve a balance between the risks and benefits inherent in any medical or surgical intervention. As we move forward with our clinical trial, we must strive to achieve this same balance, by weighing the potential benefits of our new technology against any potential risks and then perform well-designed, carefully controlled, and properly regulated investigations. This will enable us to safely bring novel techniques to the clinic where they can be used to help our patients and advance our discipline. When we first presented our work to the FDA, they were quite concerned with our seeding method. They had significant reservations about manually seeding cells onto the scaffold because of the risk of operator variability. Ultimately, we were asked to create an operator-independent method for seeding cells onto a scaffold. We developed a method for seeding the cells onto the scaffold using vacuum seeding. We designed a wand over which the scaffold could be placed. The scaffold and wand could then be inserted into a concentrated cell suspension and a vacuum applied. The cell solution is drawn through the wall of the scaffold, trapping the cells and seeding the scaffold. This method satisfied the FDA's requirement for an operator-independent seeding system. One drawback to our current method for assembling the TEVG is that it requires a class 10,000 clean rooms that costs millions of dollars to build and maintain. Ultimately this will limit the clinical utility of our technology. In order to improve our clinical utility we set out to create a closed disposable system for seeding the cells onto the scaffold that would not require a clean room. Currently, we isolate bone marrow– derived mononuclear cells using density centrifugation in Ficoll but there are other methods. Using a filter, bone
C.K. Breuer marrow–derived mononuclear cells can be trapped and then eluted off of the filter. In collaboration with the Pall Corporation, which makes a mononuclear cell filter, we have developed a prototype, which currently is in the final stages of preclinical testing. This closed disposable seeding system will preclude the need for a clean room and enable the assembly of the TEVG in any operating room sterilely and reproducibly. Venous grafts for use in congenital heart surgery are useful, but there are also other needs for different types of vascular grafts. For example, arteriovenous graft for use in angioaccess or small-caliber arterial grafts for use in peripheral bypass revascularization or coronary artery bypass surgery where the performance of currently available synthetic grafts is so poor as to preclude their use. Unfortunately, if you use our current TEVG as an arterial interposition graft, it forms aneurysms that rupture in a 10-to 14-day time period. The biomechanical properties of a TEVG are dynamic. Initially they are determined by the mechanical properties of the scaffold. But over time, as the neotissue forms and the scaffold degrades, the biomechanical properties of the neotissue become more important. Ultimately, after the scaffolding is completely degraded, the extracellular matrix produced by the neotissue determines the biomechanical properties of the neovessel. To address the problem of arterial aneurysmal dilation and rupture of the TEVG, we took a biomaterials approach and simply replaced the PGA fibers with PLA fibers in order to fabricate our scaffold. The PLA fibers are similar to the PGA fibers but degrade more slowly, enabling more time for neotissue formation. This strategy worked; the resulting TEVG no longer ruptured. However, over time, we did see some aneurysmal dilation (but no rupture) when implanted as an aortic interposition graft in a mouse model. In order to address this problem, we looked into alternative methods for fabricating our scaffold. Electrospinning is a technique that involves connecting a polymeric solution to a voltage power supply. As the voltage is increased, the polymeric solution is extruded; a Taylor cone forms. As the solvent evaporates, solid polymeric fibers are left behind, which can be collected by means of electrostatic forces. When applying the electrospinning technique to our system, we collect the fibers with a rotating mandrel, thereby enabling us to create a seamless, small-diameter polymeric scaffold. Similar to previous studies, we evaluated the electrospun TEVG in vivo by implanting the graft as an aortic interposition graft in a murine model and then used microCT to monitor the graft function. Pilot studies evaluating the electrospun TEVG demonstrate that all the grafts remain patent without evidence of thromboembolic complications or stenosis. Perhaps most importantly the electrospun TEVG showed no evidence of anueurysmal dilation graft rupture. Recently, we have begun work using IPS cells. IPS stands for inducible pleuripotent cells. These cells can be obtained by performing a skin biopsy on an individual, separating and expanding the cells in culture and then genetically manipulating the cells to revert to a pleuripotent state. This technique allows for the creation of large amounts of
Tissue-engineered vascular grafts
17
Fig. 6
Colleagues and mentors.
autologous stem cells. The stem cells can then be induced to differentiate down new lineages such as cardiomyocytes. The cardiomyocytes can then be purified and seeded onto a scaffold. Is this starting to sound familiar? It is fun to think about this sort of stuff. It's even more fun doing it! I would like to acknowledge my funding sources and give special thanks to the APSA foundation, the source of my first research grant. I would like to express my deep appreciation and gratitude to my many mentors and colleagues for their help and support over the years (Fig. 6). I would like to thank my family for their unconditional love and support. Finally and most importantly, I would ask you to wake up, the presentation is over.
Acknowledgment The author wishes to acknowledge the following funding sources: American Pediatric Surgical Association Foundation Enrichment Grant, American Surgical Association Foundation Fellowship Research Award, NIH K08HL083980, NIH R01-HL098228.
References [1] Brennan MP, Dardik A, Hibino N, et al. Tissue engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model. Ann Surg 2008;248(3):370-7. [2] Shinoka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonary artery. N Engl J Med 2001;344:532-3. [3] Hibino N, McGillicuddy E, Matsumura G, et al. Late-term results of tissue engineered vascular grafts in humans. J Thorac Cardiovasc Surg 2010;139(2):431-6. [4] Giannico S, Hammad F, Amodeo A, et al. Clinical outcome of 193 extracardiac Fontan Patients: the first 15 years. J Am Coll Cardiol 2006;47(10):2065-73. [5] Lee C, Lee CH, Hwang SW, et al. Midterm follow-up of the status of Gore-Tex graft after extracardiac conduit Fontan procedure. Eur J Cardiothorac Surg 2007;31(6):1008-12. [6] Roh JD, Nelson GN, Brennan MP, et al. Small diameter biodegradable scaffolds for vascular tissue engineering in the mouse model. Biomaterials 2007;29(10):1454-63. [7] Roh JD, Sawh-Martinez R, Brennan MP, et al. Tissue-engineered vascular grafts transform into mature blood vessels via an inflammationmediated process of vascular remodeling. Proc Natl Acad Sci 2010;107 (10):4669-74. [8] Simmon P, Kasimir MT, Seebacher G, et al. Early failure of the tissue engineered porcine heart valve Synergraft in pediatric patients. Eur J Cardiothorac Surg 2003;23:1002-6.
www.elsevier.com/locate/jpedsurg
Jay and Margie Grosfeld Lecture
The development and translation of the tissue-engineered vascular graft Christopher K. Breuer Yale University, New Haven, CT 06520, USA Received 27 August 2010; accepted 30 September 2010
Key words: Tissue-engineered vascular grafts; Development; Translation
Abstract This lecture will define the classic tissue engineering paradigm, describe cell trafficking with regard to neotissue formation, and explain the role of the host in neotissue formation. © 2011 Elsevier Inc. All rights reserved.
Dr Georgeson, thank you for the introduction, and congratulations on your APSA presidency. It is well deserved. You are a great leader in our field, a pioneer, and definitely not a cowboy or desperado.
Funding sources: American Pediatric Surgical Association Foundation Enrichment Grant American Surgical Association Foundation Fellowship Research Award NIH K08-HL083980 NIH R01-HL098228. E-mail address: [email protected]. 0022-3468/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.09.058
Dr and Mrs Grosfeld, I wanted to thank you for your continued support of APSA. Dr Grosfeld, you are the consummate pediatric surgeon and a role model for us all. I owe you a personal debt of gratitude for your support of my research through your work in the American Surgical Association and American Pediatric Surgical Association foundations. Continued support by philanthropic organizations such as the APSA foundation is critical to the continued development of our next generation of surgeon-scientists. In 1993, I was a surgical research fellow in Jay Vacanti's tissue engineering laboratory (Fig. 1). He and Dr Bob Langer were pioneers in a field that would come to be known as regenerative medicine. They had come up with the notion of creating tissue from its cellular components by seeding cells onto a biodegradable scaffold. The resulting neotissue could be used for a variety of reconstructive surgical applications. It was a magical time in the development of the field of tissue engineering, which seemed capable of blurring the boundaries between science and science fiction. The human ear on the back of the mouse (or the auriculosauris as it came to be known) became the icon for this burgeoning science. The auriculosauris project was a seminal work and proof-of-principle experiment that highlighted the immense potential of this field.
Tissue-engineered vascular grafts
9
Fig. 1
Vacanti Laboratory circa 1995.
While I had absolutely nothing to do with the design or performance of this elegant experiment, I was the first to recognize that these animals were deaf and could not hear a thing with their tissue-engineered ears. However, since I am an innovator, I recognized there were other uses for this model. The potential was enormous. I am humbled when I think of how far the field of earring design has come due to the development of this technology (Fig. 2). On a more serious note, why do research? To be completely honest with you, I went into the lab for all of the wrong reasons, as a means to an end. I wanted to be a pediatric surgeon and it had been explained to me in no uncertain terms that performing
Fig. 2
Auriculosauris with earring.
a productive research fellowship was an unwritten prerequisite, a hurdle if you will. Fortunately for me, before working in the lab, I took some time off from my general surgical residency to do some additional surgical training in order to be certain of my career choice. I worked as a junior resident on the pediatric surgical service at the Children's Hospital in Boston. Mike Caty was the chief resident and Steve Fishman the junior fellow. During this time I discovered that I loved pediatric surgery. I enjoyed working with children and their families and I especially liked operating on sick kids and making them better. However, I was equally frustrated by our inability to care for a number of children with a variety of serious surgical conditions for which there were no cures. I was particularly touched by a child who developed short bowel syndrome after undergoing an extensive bowel resection for necrotizing enterocolitis. I watched in horror as this child died an agonizingly slow death and was equally distraught to watch the devastating effect it had on her family. This experience led me to the conclusion that for me, it wasn't enough to take care of my patients. I needed to do something to advance the field and help improve our ability to treat diseases for which there were no cures. Ultimately this led me to consider work in translational research. I started in the lab with an abundance of enthusiasm and essentially no skill. For my project I had decided to follow in the footsteps of Greg Organ, the research fellow who preceded me in the Vacanti lab, and a future pediatric surgeon. I wanted to try to build upon his work tissueengineering intestine. Greg had performed some of the
10 foundational work in the field. He developed methods for isolating and culturing enterocytes, in addition to seeding and implanting tissue-engineered constructs in vivo. It was a perfect solution for my desire to advance the field through translational research. Unfortunately, after completing my first year in the laboratory, I had absolutely nothing to show for my work. I had not generated a single piece of usable data. One possible explanation was that Greg was simply wrong and that his idea would never work. A more likely explanation was simply that I wasn't a very good scientist. A partial listing of the 107 articles that both build upon or substantially advance Greg's work give credence to the notion that I wasn't a very good researcher. And so I learned the first and most important lesson of research: embrace failure. Whenever a candidate inquires about working in my lab, I always explain to them that I will teach them to fail spectacularly. It is through failure that we learn our greatest lessons and make our best discoveries. Failure, or perhaps more precisely fear of failure, fuels creativity, a scientist's best and most important attribute. After my first year in the lab, I went back to the drawing board. I made 2 lists; the first described what we could not do in the field of tissue engineering in 1994. We couldn't create capillary networks de novo, we couldn't innervate neotissue, and we couldn't grow many of the different cell types needed to make many of the organs we were interested in making. I thought I might consider working on a different project, but what to do? Upon further reflection I started to think about what we could do so I made a second list. We were really good at making ears, or other structural tissues made of bone or cartilage. We had fairly sophisticated methods for fabricating scaffolds. And while we couldn't grow a number of different cell types, even I could grow fibroblasts (the dandelion of the cell world), smooth muscle cells weren't that hard either, and since our lab was in close proximity to Judah Folkman's lab, endothelial cells practically walked up the stairs and into our incubators. If I couldn't grow intestine perhaps there was a more appropriate project.
1. Tissue engineering part II: cardiovascular tissue engineering After some thought, I concluded that I could try making tissue-engineered heart valves and blood vessels. Surprisingly this time my research worked. Here is a photo of the first tissueengineered heart valve that I made and implanted in a lamb animal model (Fig. 3). And so I learned the second lesson of research: be open to serendipity. Or as my aunt once told me, “sometimes when God closes doors He opens windows.” The development of a man-made vascular graft with growth potential would have important implications for neonates and children. Within the field of pediatric surgery, there are several important situations in which the ability to tissue engineer a blood vessel would have great utility. However, from a public health perspective this is not a major problem. Congenital heart
C.K. Breuer
Fig. 3
Tissue-engineered heart valve leaflet.
disease on the other hand is an enormous public health issue affecting nearly 1% of all live births. Despite significant advances in the treatment of congenital heart disease, it remains a leading cause of death in the newborn period. I became particularly interested in a group of diverse structural anomalies that result in a severe form of congenital heart disease that results in single-ventricle physiology. Without surgical intervention, single-ventricle cardiac anomalies are associated with 70% mortality in the first year of life while survival to adulthood is rare. Fortunately, due to significant advances in the field of congenital heart surgery, patients born with single-ventricle cardiac anomalies have markedly improved longevity and improved quality of life. In order to fix patients born with single-ventricle anomalies the surgeon must perform a series of staged operations in which the plumbing is rearranged in such a way so that the single functional ventricle pumps oxygenated blood to the body where the oxygen is delivered and then the deoxygenated blood is returned directly to the pulmonary artery where it passively passes through the pulmonary circulation and is reoxygenated. This operation is referred to as a Fontan operation. Unfortunately, like most congenital heart operations, it requires the need for synthetic grafts or patches in order to complete the operation. Complications arising from the use of synthetic grafts are a leading cause of postoperative morbidity and mortality. Synthetic grafts are a significant source of thromboembolic complications, they have poor durability due to neointimal hyperplasia and ectopic calcification, they are susceptible to infections, and they lack growth potential, which is particularly problematic in children. Occasionally, a child is born with a single-ventricle anomaly that is amenable to primary repair without the use of a synthetic conduit. This small subset of patients does quite well and has been the impetus for our work. My research focuses on the creation of tissue-engineered vascular grafts that can be created by seeding an individual's own cells onto a biodegradable scaffold. The scaffold provides sites for cell attachment and Three-dimensional space for cell growth. As the neotissue forms the scaffolding degrades ultimately creating a neovessel without any synthetic components.
Tissue-engineered vascular grafts Our scaffold is fabricated from polyglycolic acid fibers, which are woven into a tube, and coated with a 50:50 copolymer of polylactic acid and polycaprolactone. It degrades by hydrolysis losing its biomechanical integrity in 8 weeks. Total fiber degradation takes approximately 6 months. Our cell source is autologous bone marrow–derived mononuclear cells obtained by performing a bone marrow aspirate and separating the bone marrow–derived mononuclear cell fraction using density centrifugation in Ficoll. It might seem strange that we make blood vessels from bone marrow. Originally we didn't. We previously made tissue-engineered vascular grafts by performing a biopsy of an individual's own blood vessel and then isolated the endothelial cells, smooth muscle cells, and fibroblasts which we then expanded in culture. When we had grown enough cells, we seeded our scaffolds and let the seeded scaffold grow in an incubator to allow time for cell attachment. Using this technique we could make a tissue-engineered vascular graft in 2 to 3 months. This long period of time limited the clinical utility of this technique. Additionally, prolonged periods of cell culture increased the chance of contamination or even malignant dedifferentiation of the cells. We also discovered that sick people have sick cells that sometimes couldn't be grown in culture. So we began to explore alternative cell sources and came upon bone marrow– derived mononuclear cells. These cells were available in great abundance so as to preclude the need for cell culture, dramatically reducing the time it took us to create a tissueengineered vascular graft from several months to several hours. We seed a concentrated cell suspension onto the scaffold and incubate the seeded scaffold in autologous serum (also obtained from the bone marrow aspirate) for 2 hours in order to allow for cell attachment. We have studied our tissue-engineered vascular graft in a variety of animal models. In order to evaluate the growth potential of the tissue-engineered vascular graft, we inserted it as an intrathoracic IVC interposition graft in a juvenile lamb model. The IVC replacement model is a validated animal model for investigating the use of grafts in the high-flow, lowpressure circulatory systems such as the Fontan circulation. The lamb model is the Food and Drug Administration's preferred model for investigating biological vascular grafts for use in congenital heart surgery due to the lamb's propensity for accelerated ectopic calcification, which is the leading cause of failure for biological conduits used in the repair of congenital cardiac anomalies. Next we serially monitored the tissueengineered vascular grafts using MRI. Using computer generated 3-dimensional imaging, we were able to superimpose images of the tissue-engineered vascular grafts at varying time points and thereby showed that the tissue-engineered vascular grafts increased in size over time. The increase in size was symmetrical and proportional to the increase in size of the surrounding vasculature suggesting growth instead of aneurysmal dilation [1]. In 2003, I came to Yale University as part of its tissue engineering initiative. Yale is a fairly small institution and compensates for its size by recruiting faculty in areas of
11 thematic research so as to optimize and build upon potential synergies. I have been repeatedly instructed by my new mentors to focus. I was told if I truly wanted to advance the field I would need to dig deeper and become more specialized. I needed to develop expertise in vascular biology, biomedical engineering, in addition to surgery. And so I learned the third lesson of research: Focus, focus, focus. The corollary to “focus, focus, focus” is that multidisciplinary collaborations are critical for success particularly for complex translational research projects. This requires certain leadership skills for it is akin to herding cats. We began to further characterize the structure of our tissue-engineered vascular grafts. We used histological stains and immunohistochemical techniques to demonstrate that the neovessels that resulted from the tissue-engineered vascular grafts were comprised of a monolayer of endothelial cells lining the luminal surface of the vessel surrounded by concentric layers of smooth muscle cells embedded in an extracellular matrix rich in both collagen and elastin similar to the native IVC. Neovessels even express Eph B4 the molecular determinant of venous development. During embryology a blood vessel is not considered a vein until Eph B4 is expressed. These findings suggest that neovessels are truly veins and not simply viable conduits. In 2001, Toshi Shinoka, my lab partner and the individual with whom I developed the basic techniques for tissueengineering blood vessel, reported the first clinical application of vascular tissue engineering [2]. He used a patch to perform a pulmonary artery angioplasty in a child undergoing an operation for pulmonary artery stenosis. He subsequently
Fig. 4 Three-dimensional CT of a tissue-engineered vascular graft 3 years after implantation. Reprinted from J Thorac Cardiovasc Surg 139(2) Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. 431-6 (2010).
12
C.K. Breuer
performed the first pilot study investigating the use of tissueengineered vascular grafts in congenital heart surgery. Here we see an image of a tissue-engineered vascular graft 3 years after implantation demonstrating a widely patent conduit (Fig. 4). In 2007, I was fortunate to be reunited with Dr Shinoka at Yale where he was recruited to be the head of the congenital heart surgery program. This highlights the fourth lesson of research: surround yourself with good people. Good not only in the sense of capable, but more importantly, good in the sense of a person with similar values, a strong work ethic, and complementary goals. This also applies to the selection of people who work for you. I have been fortunate to select research fellows who are uniformly excellent and are a real source of pride for me and make coming to work a pleasure. For a physician scientist it is also critical to surround yourself with great partners who understand and appreciate the role of research in medicine. With Dr Shinoka on board, we decided to compile all of our preclinical and clinical data in order to submit an application to the FDA for the first clinical trial in the United States evaluating the use of tissue-engineered vascular grafts in humans. This table summarizes the early (1-year) follow-up on 25 patients who had undergone extracardiac total cavopulmonary connection (EC TCPC) modified Fontan surgery using
Table 1
a tissue-engineered vascular graft (Table 1). There were no graft-related deaths and all tissue-engineered vascular grafts were patent and intact. The only complication was a partial mural thrombus that was successfully treated with coumadin. This table summarized the late (5- to 7-year follow-up) on this cohort of patients (Table 2). Again there was no graftrelated mortality. All the tissue-engineered vascular grafts were patent and intact. There were no new thromboembolic complications; however, 4 patients developed stenosis requiring angioplasty. This raises the obvious question: What is the rate of stenosis for PTFE grafts (the most commonly used grafts in Fontan surgery)? The incidence of stenosis in this cohort is poorly studied and difficult to ascertain from the literature. As in our series, EC TCPC conduit stenosis is typically asymptomatic and will not be detected unless the patient is imaged, which is not routinely done in this patient population. This results in underreporting. All patients in our study were routinely imaged as part of the investigative protocol. There are only 2 papers in the literature that serially monitor EC TCPC conduits using the same imaging modality [4,5]. The first is a large series from Italy, which selectively and randomly imaged a fraction of their cohort using serial MRI. They noted an 18% to 32% narrowing of the conduit within the
Patient status 1 year after TEVG implantation
Patient
Age at operation (y)
Graft type
Graft size (cm)
Patient status
Graft status
Graft patency
Graft-related complications
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
2 1 7 21 4 12 17 19 3 2 2 13 2 2 2 2 24 1 11 2 3 4 4 13 2
PLA PLA PLA PLA PLA PLA PLA PLA PLA PLA PGA PLA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA
16 20 18 24 20 24 24 22 12 16 16 20 16 18 12 16 18 16 18 16 16 18 18 16 18
Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive
Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact
Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent
None None None None None None None None None None None None Thrombosis None None None None None None None None None None None None
Early results of clinical trial [3]. TEVG indicates tissue-engineered vascular graft; PLA, poly-L-lactic acid: PGA, polyglycolic acid. Reprinted from J Thorac Cardiovasc Surg 139(2) Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. 431-6 (2010).
Tissue-engineered vascular grafts Table 2
13
Patient late-term status after TEVG implantation
Patient
Age of operation
Graft type
Graft size (cm)
Patient status
Graft status
Graft patency
Graft-related complications
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
2 1 7 21 4 12 17 19 3 2 2 13 2 2 2 2 24 1 11 2 3 4 4 13 2
PLA PLA PLA PLA PLA PLA PLA PLA PLA PLA PLA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA PGA
16 20 18 24 20 24 24 22 12 16 20 16 16 18 12 16 18 16 18 16 16 18 18 16 18
Alive Alive Alive Alive Alive Alive Alive Dead Alive Dead Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead
Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact
Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent Patent
None None Stenosis a None None None None None Stenosis a None None Stenosis a Thrombosis None None None None Stenosis a None None None None None None None
Late results of clinical trial [3]. TEVG indicates tissue-engineered vascular graft; PLA, poly-L-lactic acid; PGA, polyglycolic acid. Reprinted from J Thorac Cardiovasc Surg 139(2) Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. 431-6 (2010). a This patient underwent successful angioplasty.
first 6 months after implantation, which tended to stabilize and not to progress in most conduits after this time point [4]. The second paper was from a large Korean cohort, which was serially monitored using serial angiography. This group noted similar findings demonstrating around a 20% narrowing after the first 6 months after implantation [5]. Furthermore, the optimal treatment for asymptomatic stenosis has yet to be determined. In some studies, stenotic EC TCPC conduits were selectively treated with angioplasty; however, most EC TCPC conduit stenosis is treated with surgical graft replacement, which is associated with a 3% to 5% mortality. All 4 of our patients who developed stenosis were successfully treated with angioplasty or angioplasty and stenting. Overall these results compare favorably with synthetic vascular grafts. Synthetic vascular grafts appear to have a higher incidence of thromboembolic complications, while tissue-engineered vascular grafts have a higher incidence of stenosis. Tissue-engineered vascular grafts, however, are the only man-made grafts with growth potential. These clinical data have proven useful in directing our research efforts. In an attempt to rationally design an improved second-generation tissue-engineered vascular graft, we are currently investigating the mechanisms underlying tissue-engineered vascular graft stenosis.
Data obtained using large animal models provided us with some insights into the process of neovessel formation. Evaluation of histological specimen obtained over a 6-month time course demonstrated that neotissue forms both in and on the scaffold. Over time the scaffold degrades and the layers fuse and mature creating a laminated structure possessing a neointima, neomedia, and neoadeventitia. Understanding what factors control this process and which factors are important in the formation of stenosis is critical to our work. Over the past 5 years my laboratory has devoted significant effort toward developing a mouse model to study neovessel formation and tissue-engineered vascular graft stenosis. It may seem counterintuitive to develop a mouse model after having already translated our work from the bench to the clinic; however, there are a large number of molecular reagents that are readily available only for mouse work. These reagents are critical to this sort of work. Additionally, the use of a murine model has the added benefit of being able to perform experiments more efficiently and more cost effectively. To put this in perspective, in our first 10 years of research, we performed less than 200 implants; we currently perform over 500 per year. This allows us to ask and answer questions very rapidly. As a first step in developing a mouse model, we needed to create a method for fabricating the scaffold on a much smaller
14 scale. Jason Roh, a Yale medical student, developed a method for making the tissue-engineering scaffolds using the same design and materials as in the clinical trial but on a much smaller scale. The resulting scaffolds have the same porosity and degradation properties as the scaffolds used in the clinical trial but with diameters between 500 and 1000 μm, which enable implantation into the mouse vasculature. Using microsurgical technique, the tissue-engineered vascular graft can be implanted as an IVC interposition graft. We can then use micro-CT angiography to monitor neovessel formation. We have demonstrated that the tissue-engineered vascular grafts undergo the same remodeling over time that we have seen in large animal models and that the resulting neovessel resembles the native IVC both histologically and grossly. Once again, using immunohistochemical staining techniques, we have demonstrated that the neovessel is composed of a monolayer of endothelial cells lining the luminal surface surrounded by concentric layers of smooth muscle cells embedded in an exrtracellular matrix similar to that of the native IVC [6]. We have used time-course studies to break this process into phases. In the first phase, mononuclear cells invade the scaffold-forming macrophages. The concentration of macrophages in the scaffold initially increases. Endothelial cells and smooth muscle cells begin to form a neointima and neomedia on the inner surface of the scaffold. The scaffold itself gives rise to the neoadventitia. As the scaffolding degrades the vascular neotissue remodels, ultimately forming a neovessel that resembles the native IVC [7]. The first question we attempted to answer using the murine model is what is the role of cell seeding? Using microCT angiography we were able to demonstrate that seeded TEVG remained widely patent while unseeded TEVG tended to fail. The TEVG occlusion developed gradually as demonstrated by the formation of collateral blood vessels. Histological evaluation of the TEVG demonstrated that the mechanism of graft failure is stenosis. Thus, cell seeding improves patency by decreasing the incidence of stenosis. The next question we asked is what is the identity of the seeded cells? Bone marrow–derived mononuclear cells are composed of a heterogeneous population of mononuclear cells. We used multicolor florescent activated cell sorting to identify various subpopulations of cells within the bone marrow–derived mononuclear cell population. We had always assumed that the bone marrow–derived mononuclear cells were a rich source of vascular stem cells such as endothelial progenitor cells or mesenchymal stem cells; however, when we quantified the actual number of stem cells in the bone marrow–derived mononuclear cell population, it became obvious to us that thus was not the case. When we calculated the number of stem cells seeded onto the scaffold, we noted that only a few of these stem cells were attached to any single tissue-engineering scaffold [7]. What is the fate of the seeded cells? In order to determine the fate of the seeded cells, we created a chimera by seeding human bone marrow–derived mononuclear cells onto the tissue-engineering scaffold and implanted the human TEVG
C.K. Breuer into an immunosuppressed SCID-beige mouse. SCID-beige mice lack an adaptive immune response and readily tolerate xenotransplantation. Next we harvested the TEVG over a time course and tracked the seeded cells using human-specific immunohistochemical stains. We demonstrated that the human cells disappeared within 3 to 7 days after implantation. These findings flew in the face of the classic tissue-engineering paradigm in which the seeded cells are viewed as the building blocks of neotissue and were quite a surprise. We validated these findings using human-specific molecular probes, again showing that the seeded cells disappear. What are the cellular components of vascular neotissue? Since the seeded cells were not the building blocks of the vascular neotissue, what were the cells? Immunohistochemical analysis demonstrated that during the early phase of neotissue formation the vast majority of cells were host-derived macrophages. Quantitative analysis of the macrophage infiltrate demonstrated that cell seeding affected the degree of macrophage infiltration and that the degree of macrophage infiltration also correlated with the degree of stenosis suggesting that the host-derived macrophages may play a role in neovessel formation and the development of tissue-engineered vascular graft stenosis. Thus, we concluded that neovessel formation and the development of tissue-engineered vascular graft stenosis are inflammation-mediated processes. What are the molecular mechanisms of neovessel formation? How do the seeded cells exert such a powerful biological effect if they disappear within a few days after implantation? One possible explanation is that they exert a paracrine effect by initiating a cascade of events that ultimately lead to neovessel formation. In order to identify candidate cytokines important in this process, we measured cytokine production by the seeded tissue-engineered vascular grafts in vitro. We noted that the seeded cells produced relatively large quantities of MCP-1 (monocyte chemoattractive protein-1), a powerful chemokine that attracts macrophages wherever it is released. We hypothesized that this may play an important role in neovessel formation and the development of tissue-engineered vascular graft stenosis. In order to determine if MCP-1 production by the seeded cells was critical to neovessel formation and the development of tissue-engineered vascular graft stenosis, we took advantage of the fact that we had developed a mouse model and could therefore use transgenics. Transgenic mice are genetically engineered and bred to have specific genetic abnormalities. Knockout mice lack a specific gene. In the case of a MCP-1 knockout or null mouse, the MCP-1 gene is missing from every cell in the animal's body. Wild type refers to control mice, which are syngeneic with the exception of the knockout gene, which is present. We isolated bone marrow–derived mononuclear cells from either MCP-1 knockout mice or wild-type mice and used them to make TEVG, which were implanted into SCID-beige mice and harvested over a time course. Morphometric evaluation of the grafts demonstrated that the TEVG created using the MCP-1 knockout cells had narrower lumens and behaved
Tissue-engineered vascular grafts more like unseeded TEVG and less like seeded TEVG, suggesting MCP-1 was necessary for the process of neotissue formation and the development of TEVG stenosis. In order to see if MCP-1 was sufficient for the process of neovessel formation and the development of TEVG stenosis, we used controlled release technology to crate MCP-1 eluting microspheres. A microsphere is a microscopic particle created from a biodegradable material. A protein (in this case MCP-1) could be encapsuled in the microsphere. When encapsulated, the protein is in a state of suspended animation, but when released, it can exert its biologic effect and be degraded. We then incorporated the MCP-1 eluting microspheres into the design of our tissue-engineering scaffold creating an MCP-1 eluting tissue-engineered vascular graft. The MCP-1 eluting tissueengineering scaffold eluted MCP-1 at a rate and duration similar to the amount and duration of MCP-1 production by the seeded cells. Morphometric evaluation of the biomimetic TEVG demonstrated that the MCP-1 eluting scaffolds had larger diameters similar to the seeded TEVG and less like the unseeded TEVG. These findings suggested that MCP-1 is a critical player in neovessel formation. We currently are investigating the roles of other cytokines including PDGF and VEGF in this process in an attempt to better understand the mechanisms of neovessel formation and the development of TEVG stenosis. In our current model of neovessel formation, we believe that the seeded bone marrow–derived mononuclear cells release MCP-1 that attracts monocytes from the circulation into the scaffold where they become macrophages. The macrophages, in turn, release PDGF and VEGF which induce in-growth of the neighboring smooth muscle cells and endothelial cells. As the scaffold degrades, the macrophages begin to disappear and the vascular neotissue forms continuously remodeling until the scaffolding is completely gone and the neovessel is formed. Thus, at least in our model system, we have disproven the notion that the neovessel is formed from the seeded cells as originally proposed
Fig. 5
15 according to the classic tissue engineering paradigm in which the seeded cells are viewed as the building blocks of neotissue. Instead, these findings suggest that the mechanism of neovessel formation is regeneration of the blood vessel sort of like a salamander regrows its tail. This sort of mechanistic understanding has important implications for the rationale design of improved second generation TEVG. We recently submitted an application to the FDA supporting the performance of the first clinical trial in the United States investigating the use of TEVG in humans. We hope to bridge the chasm between the research and clinical practice and hopefully improve our ability to care for our patients. When I first began this project I assumed that there was a streamlined process with instructions that enabled a seamless transition from the bench to the clinic. Nothing could have been farther from the truth. This diagram outlines the path typically taken in order to bring a product from the bench to the clinic (Fig. 5). What had never been explained to me was that this is all considered proprietary information, which companies typically will not share. To make matters worse, there were no FDA-approved TEVG, so there was no president upon which to build. We needed to blaze our own trail following an extensive but vague roadmap. Four years later I am please to announce that we recently received approval from the FDA for performing our study. Obtaining approval was a long and arduous process; a single copy of our application was over 3000 pages. And so I learned the fifth lesson of research: work hard. There is no substitute. As I prepare to begin our clinical investigation, I realize that my research is truly only just beginning. I think of 2 papers that have dramatically altered my career and directed my actions. The first is Toshi Shinoka's initial case report in the New England Journal of Medicine describing the first clinical use of a TEVG [2]. It's hard to describe what a wonderful feeling it is to see an idea come to clinical fruition
Regulatory pathway for medical product development and translation (adapted from FDA Web site).
16 and be used to help a patient. The second article was published in a less prestigious journal and describes the research of one of my contemporaries who designed and developed a tissue-engineered heart valve created by seeding human cells onto a decellularized porcine heart valve, essentially using the extracellular matrix as a tissueengineering scaffold [8]. The investigators had performed some very exciting preclinical work demonstrating the feasibility of this technology and went on to perform a pilot study in humans. Four of the initial 6 patients implanted with the tissue-engineered heart valve died. The remaining 2 patients had to have their tissue-engineered heart valves removed and replaced with artificial heart valves. Post hoc analysis demonstrated that the pig heart valve had not been completely decellularized. The investigators had implanted a xenograft heart valve, which had been promptly rejected. This article highlights the critical role of proper regulatory control in any study. I am reminded that the road to perdition is paved with good intentions. We have all sworn a Hippocratic oath to first do no harm. A literal interpretation of this statement would bring the practice of surgery to a screeching halt. In fact, in the original Hippocratic oath, surgery was strictly forbidden. A closer reading of the original text suggests that this is not the idea that Hippocrates was originally trying to convey. Instead he was trying to insist that it is a physician's duty to try to achieve a balance between the risks and benefits inherent in any medical or surgical intervention. As we move forward with our clinical trial, we must strive to achieve this same balance, by weighing the potential benefits of our new technology against any potential risks and then perform well-designed, carefully controlled, and properly regulated investigations. This will enable us to safely bring novel techniques to the clinic where they can be used to help our patients and advance our discipline. When we first presented our work to the FDA, they were quite concerned with our seeding method. They had significant reservations about manually seeding cells onto the scaffold because of the risk of operator variability. Ultimately, we were asked to create an operator-independent method for seeding cells onto a scaffold. We developed a method for seeding the cells onto the scaffold using vacuum seeding. We designed a wand over which the scaffold could be placed. The scaffold and wand could then be inserted into a concentrated cell suspension and a vacuum applied. The cell solution is drawn through the wall of the scaffold, trapping the cells and seeding the scaffold. This method satisfied the FDA's requirement for an operator-independent seeding system. One drawback to our current method for assembling the TEVG is that it requires a class 10,000 clean rooms that costs millions of dollars to build and maintain. Ultimately this will limit the clinical utility of our technology. In order to improve our clinical utility we set out to create a closed disposable system for seeding the cells onto the scaffold that would not require a clean room. Currently, we isolate bone marrow– derived mononuclear cells using density centrifugation in Ficoll but there are other methods. Using a filter, bone
C.K. Breuer marrow–derived mononuclear cells can be trapped and then eluted off of the filter. In collaboration with the Pall Corporation, which makes a mononuclear cell filter, we have developed a prototype, which currently is in the final stages of preclinical testing. This closed disposable seeding system will preclude the need for a clean room and enable the assembly of the TEVG in any operating room sterilely and reproducibly. Venous grafts for use in congenital heart surgery are useful, but there are also other needs for different types of vascular grafts. For example, arteriovenous graft for use in angioaccess or small-caliber arterial grafts for use in peripheral bypass revascularization or coronary artery bypass surgery where the performance of currently available synthetic grafts is so poor as to preclude their use. Unfortunately, if you use our current TEVG as an arterial interposition graft, it forms aneurysms that rupture in a 10-to 14-day time period. The biomechanical properties of a TEVG are dynamic. Initially they are determined by the mechanical properties of the scaffold. But over time, as the neotissue forms and the scaffold degrades, the biomechanical properties of the neotissue become more important. Ultimately, after the scaffolding is completely degraded, the extracellular matrix produced by the neotissue determines the biomechanical properties of the neovessel. To address the problem of arterial aneurysmal dilation and rupture of the TEVG, we took a biomaterials approach and simply replaced the PGA fibers with PLA fibers in order to fabricate our scaffold. The PLA fibers are similar to the PGA fibers but degrade more slowly, enabling more time for neotissue formation. This strategy worked; the resulting TEVG no longer ruptured. However, over time, we did see some aneurysmal dilation (but no rupture) when implanted as an aortic interposition graft in a mouse model. In order to address this problem, we looked into alternative methods for fabricating our scaffold. Electrospinning is a technique that involves connecting a polymeric solution to a voltage power supply. As the voltage is increased, the polymeric solution is extruded; a Taylor cone forms. As the solvent evaporates, solid polymeric fibers are left behind, which can be collected by means of electrostatic forces. When applying the electrospinning technique to our system, we collect the fibers with a rotating mandrel, thereby enabling us to create a seamless, small-diameter polymeric scaffold. Similar to previous studies, we evaluated the electrospun TEVG in vivo by implanting the graft as an aortic interposition graft in a murine model and then used microCT to monitor the graft function. Pilot studies evaluating the electrospun TEVG demonstrate that all the grafts remain patent without evidence of thromboembolic complications or stenosis. Perhaps most importantly the electrospun TEVG showed no evidence of anueurysmal dilation graft rupture. Recently, we have begun work using IPS cells. IPS stands for inducible pleuripotent cells. These cells can be obtained by performing a skin biopsy on an individual, separating and expanding the cells in culture and then genetically manipulating the cells to revert to a pleuripotent state. This technique allows for the creation of large amounts of
Tissue-engineered vascular grafts
17
Fig. 6
Colleagues and mentors.
autologous stem cells. The stem cells can then be induced to differentiate down new lineages such as cardiomyocytes. The cardiomyocytes can then be purified and seeded onto a scaffold. Is this starting to sound familiar? It is fun to think about this sort of stuff. It's even more fun doing it! I would like to acknowledge my funding sources and give special thanks to the APSA foundation, the source of my first research grant. I would like to express my deep appreciation and gratitude to my many mentors and colleagues for their help and support over the years (Fig. 6). I would like to thank my family for their unconditional love and support. Finally and most importantly, I would ask you to wake up, the presentation is over.
Acknowledgment The author wishes to acknowledge the following funding sources: American Pediatric Surgical Association Foundation Enrichment Grant, American Surgical Association Foundation Fellowship Research Award, NIH K08HL083980, NIH R01-HL098228.
References [1] Brennan MP, Dardik A, Hibino N, et al. Tissue engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model. Ann Surg 2008;248(3):370-7. [2] Shinoka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonary artery. N Engl J Med 2001;344:532-3. [3] Hibino N, McGillicuddy E, Matsumura G, et al. Late-term results of tissue engineered vascular grafts in humans. J Thorac Cardiovasc Surg 2010;139(2):431-6. [4] Giannico S, Hammad F, Amodeo A, et al. Clinical outcome of 193 extracardiac Fontan Patients: the first 15 years. J Am Coll Cardiol 2006;47(10):2065-73. [5] Lee C, Lee CH, Hwang SW, et al. Midterm follow-up of the status of Gore-Tex graft after extracardiac conduit Fontan procedure. Eur J Cardiothorac Surg 2007;31(6):1008-12. [6] Roh JD, Nelson GN, Brennan MP, et al. Small diameter biodegradable scaffolds for vascular tissue engineering in the mouse model. Biomaterials 2007;29(10):1454-63. [7] Roh JD, Sawh-Martinez R, Brennan MP, et al. Tissue-engineered vascular grafts transform into mature blood vessels via an inflammationmediated process of vascular remodeling. Proc Natl Acad Sci 2010;107 (10):4669-74. [8] Simmon P, Kasimir MT, Seebacher G, et al. Early failure of the tissue engineered porcine heart valve Synergraft in pediatric patients. Eur J Cardiothorac Surg 2003;23:1002-6.