The estimated prevalence of congenital cardiovascular disease in the United States ranges from 6.5 hundred thousand to 1.3 million people. Approximately 1 in every 100 infants is expected to have heart defects, totaling to about 36,000 infants per year. Ten percent of these cases—over 3,600 infants—result in death. However, with emerging technology in tissue engineered vascular grafts, patients can be afforded a much longer life expectancy.
But occasionally, due to the particular anatomy of the child, a surgeon can operate and repair the heart without using an artificial graft. In such cases, the surgeon is able to sew one blood vessel to another. Although these children usually have a higher success rate and fewer complications, such cases are only possible in about one percent of children with congenital heart disease. The idea that patients who have had their hearts mended by natural, rather than artificial, means has served as the impetus for the work of Dr. Christopher Breuer and Dr. Toshiharu Shinoka, Professors of Pediatrics at the Yale School of Medicine.
In the past, the process for constructing a tissue engineered vascular graft (TEVG) was tedious and long-winded. In the “old fashioned way,” a physician would have to harvest the patient’s cells through a blood vessel biopsy and then proceed to separate and expand the different cells in culture. This process required many weeks, as it was necessary to wait for the cells to gradually multiply to have enough cells for the graft. Some patients do not have many days to survive, let alone months to wait for a personalized vascular graft to be made. Compounding the problem was the discovery that sick people usually have sick cells, so the patients who perhaps needed the vascular grafts the most had sick cells and a TEVG could not be made.
However, Breuer and Shinoka utilized an innovative, highly efficient method for creating TEVGs. They took a biodegradable synthetic scaffold, made of the same material as absorbable sutures, and seeded the individual’s own cells onto it. The scaffold degrades by hydrolysis, ultimately leaving only the living vessel in the patient. The unique scaffold material is vital to the process because it is able to degrade in everyone and offers little variability since all people have approximately the same composition of water in their bodies.
In order to harvest the cells, they performed a bone marrow aspirate, where a needle was put through the cortex of the spinal bone and marrow. The marrow was drawn up and separated by density centrifugation. This yielded bone marrow dry mononuclear cells (BMC) that were directly seeded onto the scaffold by pipetting. The seeded scaffold was then incubated in the patient’s plasma for two hours in order for the cells to attach to the scaffold. The graft was then ready for implantation. What used to be a multiple month marathon has been reduced to a few-hour process that can produce a viable TEVG on the same day as the cell harvest.
After implanting TEVGs in lambs, Breuer and Shinoka found that their grafts were functional, actually growing in size and remodeling themselves in the host. They then investigated how the veins formed and ways to improve the vascular grafts. They created a model of the vascular graft to be used on people and made it small enough to put into a mouse. The scaffold was seeded with human BMC and implanted into an immunocompromised mouse. They proceeded to conduct a variety of tests that yielded surprising results.
When looking at the integration of tissue-engineered grafts with the human body, the conventional wisdom was that the cells seeded onto the scaffold were the building blocks of the tissue, which Breuer likens to “the bricks you made the house from.” However, they found that the BMC do not become part of the neovessel but are rapidly replaced by host cells. The seeded cells are still necessary though, as without seeding, the grafts constrict and do not allow optimal blood flow. This showed that the seeded BMCs played only an indirect role in the early stages of the vascular development.
Rather than the BMCs integrating into the host, they trigger an inflammatory mechanism initiated by the invasion of host monocytes (white blood cells) that in turn recruits more host monocytes to the scaffold. Bruer and Shinoka’s research showed that this early monocyte recruitment is deeply involved with natural blood vessel formation after birth. In particular, the monocyte recruitment is caused by the seeded BMCs secreting various cytokines or signaling proteins, such as MCP-1, which recruit the host cells to the scaffold. In a short period of time, the seeded cells disappear and the host cells become incorporated into the scaffold and graft.
Then, the host cells release a different series of cytokines, such as VEGF and PDGF, which then recruit the actual cells that compose the blood vessel—the smooth muscle cells and endothelial cells. Simultaneous to cell recruitment and tissue formation, the scaffolding degrades so that only a blood vessel remains. Thus, TEVG vessel formation is governed by a paracrine effect, where the seeded cells send a message causing a cascade of events, ultimately resulting in a blood vessel free of any artificial matter.
Initially, the host treats the scaffold as a foreign body, but as the graft degrades, the inflammatory response subsides and finally ceases. This indicates that the inflammatory response, regardless of its initial function, is innocuous to the host. Furthermore, with time, the vascular graft is recognized as an actual vein. A blood vessel cannot be considered a vein or artery until the protein FB-4 or FB-2, respectively, is expressed. The researchers looked at the production of these proteins in the grafts and found that while the grafts originally do not possess them, they do express the proteins after the scaffolding is gone. This further supports the view that the neovessels are actually veins.
TEVG vs. Artificial Grafts
Tissue engineered vascular grafts show promise for those who with heart defects, but how do they compare with the synthetic grafts? In Toshiharu’s 2001 study in Japan, twenty-five TEVGs were implanted in patients between the ages of five and seven years who had single ventricle physiology and the patients were followed. The results were promising, with no graft-related mortality and no evidence of major structural malformations. The only problem encountered was graft stenosis, or constricting, which was easily treated by an angioplasty, a procedure to widen the narrowed blood vessel. In comparison to synthetic grafts, if an artificial graft develops a stenosis, an operation to replace the entire segment is needed, as it does not naturally grow with the body. In contrast, the TEVGs are living grafts that are incorporated into the body, as they can grow, repair, and remodel, whereas artificial grafts are susceptible to infection, poor durability, and calcification when being attacked by the immune system. However, TEVGs are an encouraging alternative to these problems.
Looking forward, tissue engineered vascular grafts can be applied to the whole vascular system. Though they have not been done on humans, Breuer and Shinoka have implemented many grafts in animals and used them for a multitude of applications, such as arterial grafts and arterial venous graft for dialysis. Breuer says, “The hierarchy is to go from venous grafts to arterial venous grafts, to arterial grafts to small arterial grafts, and then valve conduits where you make blood vessels attached to heart valves.” With respect to the application of these grafts, venous grafts are not widely used in surgery but are used in congenital heart surgery. Arterial grafts, on the other hand, are widely used by the almost 500,000 patients a year in the United States who need arterial bypasses. Also, as our population gets older, heart failure and the need for heart transplants will increase. However, there is a limited number of hearts for transplantation and even those available are not an exact match for one’s own immune system. In light of this, Breuer and Shinoka are trying to make heart tissue and a fully functioning heart. Though it is “a whole order of magnitude tougher” due to the complex capillary network that needs to be formed, it is a step in the right direction.
Breuer and Shinoka have a few take-away messages from their work. First, their research is ongoing. Their patients are their number one priority and after investing many years of hard work, they are “just getting to the point where we’re studying [TEVGs] still.” Breuer remarks, “If you really want to make a difference and get something to a clinic and help a patient, you have to take one thing at a time and see it through.” Next, they are doing translation research where they are not only studying things in the lab but are actively bringing their research to the clinic. They will soon conduct a human study for TEVG, marking the first time that there has been a FDA approved human study for TEVG in the world.
After fifteen years, Dr. Christopher Breuer and Dr. Toshiharu Shinoka have developed a deep understanding of the mechanisms and the applications of tissue engineered vascular grafts in humans. TEVGs provide a promising future on multiple frontiers. With a clinical trial upcoming, we can only hope that these two researchers uncover more promising information that will allow present and future generations to have much happier, healthier hearts.
About the author: Sudhakar Nuti is a freshman in Trumbull College. He is a prospective Classics major who is infatuated with science.
Acknowledgements: I would like to thank Dr. Christopher Breuer and Dr. Toshiharu Shinoka for all their time and help. I wish them the best in their future studies and look forward to what they discover next.
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