Tissue from the lab mends a broken heart

Angela Irizarry was still in her mother’s womb when tests revealed that one of her heart’s two ventricles wasn’t working. She was destined to be a “blue baby.” With only one functional ventricle, oxygenated and deoxygenated blood would mix within that ventricle, causing hypoxia and the bluish coloration of the skin that gives the syndrome its name. In 1968 a surgeon named Francis Fontan, M.D., devised a procedure that has become the standard of care to palliate this condition. It involves three surgeries to redirect blood flow: one a few days after birth; a second a few months later; and a third when the child reaches the age of three. In that final procedure a plastic tube is inserted to channel blood from the inferior vena cava to the lungs. For Angela, however, surgeons had something different in mind. She would undergo the first two surgeries as usual—but for the third, a biodegradable scaffolding seeded with cells from her own bone marrow would replace the plastic tube. She would be the first patient in the United States to receive a bioengineered cardiac blood vessel.

A meeting at MIT

On the window sill of his office in the Boardman Building, Toshiharu Shinoka, M.D., Ph.D., keeps a photo of his days as a postdoctoral fellow in Boston in the 1990s, where he met his friend and colleague, Christopher K. Breuer, M.D. Also in the group photo are Joseph P. Vacanti, M.D., an expert in tissue engineering; and Robert Langer, Ph.D., the MIT professor renowned for his innovative medical devices. Shinoka, associate professor of surgery and pediatrics and director of pediatric cardiovascular surgery, and Breuer, associate professor of pediatric surgery and pediatrics, met while studying under Vacanti and Langer at Children’s Hospital Boston.

“What made me become interested in tissue engineering was the fact that on a daily basis in my training I would see problems arise that we couldn’t fix. I would watch children die and watch families fall apart. That obviously left a bad taste in my mouth,” said Breuer. “In surgery there are many problems we could fix if we had adequate tissue. Tissue engineering provided means of creating an abundant source of tissue for surgical reconstructive procedures.”

The pair first attempted to engineer intestines, but found that the science of the day was not up to the task of producing such complex tissue. They switched to blood vessels and found a technique that worked. They created a biodegradable scaffolding with a matrix for seeding cells as well as sites for cell attachment and tissue formation, then implanted it in a lamb. “The scaffolding degrades and the remaining tissue is viable and it’s made from people’s own tissue, so you can avoid problems like rejection,” Breuer said.

But problems remained. “Using our original method it took three months to grow the cells, and seed, and incubate the scaffold. Surgeons don’t have three months to wait for a blood vessel,” Breuer said. Waiting that long also increased the odds of contamination and the risk of evolution running its course—cells in culture could differentiate and change their identity or even become tumor cells.

The collaboration ended when Breuer entered the U.S. Air Force and Shinoka returned to Tokyo Women’s Medical University Hospital to continue his research. He switched from blood vessel cells to bone marrow cells, which are so abundant that they don’t need to be grown in culture. That cut the time needed to make the grafts from a few months to a few hours. Shinoka began implanting the biodegradable grafts in humans with promising results.

When Breuer heard of Shinoka’s success he was still in the military and attending to injured veterans of the war in Afghanistan. “It was one of those days in your life where you get hit by lightning,” Breuer said. “I just knew what I was going to do with the rest of my career.”

By 2003 Breuer had moved to Yale, lured by a new tissue engineering initiative and faculty, including W. Mark Saltzman, Ph.D., chair and Goizueta Foundation Professor of Biomedical Engineering and Chemical and Environmental Engineering, who had also trained under Langer at MIT; Jordan S. Pober, M.D. ’77, Ph.D. ’77, HS ’78, Ensign Professor of Immunobiology, professor of dermatology and of pathology, and director of the Human and Translational Immunology Program; and William C. Sessa, Ph.D., Alfred Gilman Professor of Pharmacology and director of the Vascular Biology and Therapeutics Program. “It was a wonderful environment in which to perform translational research,” Breuer said.

Three years later he urged Shinoka to join him. “It was very suitable for our future research so I decided to come over here,” Shinoka said. “In the United States no one was doing this kind of approach.”

Rearranging the heart

“When I was five months pregnant they asked me to have a test because I was 37 years old and there was a risk that the baby would have defects,” recalled Claudia Irizarry, Angela’s mother. She works as a secretary in a church; her husband, Angel, is a contractor. “They figured out that all her organs were backwards. In the same ultrasound they saw that she had this heart condition. Right away they told me that she was going to need three surgeries.”

Congenital heart disease is a common birth defect, occurring in one in 100 live births. Single-ventricle physiology, a relatively uncommon subcategory of congenital heart disease, stems from several heart anomalies but leaves children with only one functional ventricle. One of the two ventricles may be larger than the other, or there may be no wall between the two ventricles. These defects force the functioning ventricle to do the work of two, pumping blood for both the lungs and the rest of the body. It also mixes oxygen-rich blood leaving the lungs with deoxygenated blood from the veins from the lower body, leading to the “blue baby” syndrome. Without surgical intervention, about 70 percent of children with the defect die in the first year of life. Few reach adulthood. The Fontan procedure, though palliative rather than curative, has been the only solution.

“What this operation did was rearrange the plumbing, so that that one ventricle could pump blood to the body, and then that blood would return and go through the pulmonary circulation passively, without a ventricle pushing it through. This prevented the mixing of blood so the children were no longer cyanotic,” Breuer said.

Despite its benefits, the Fontan procedure posed problems. It required the use of synthetic materials that could lead to blood clots and other complications. It also meant a lifetime of blood-thinning drugs for the patients. The synthetic grafts left children vulnerable to infections. And the grafts could not grow with the children. “Children can outgrow their operations, just the way they can outgrow their shoes. Imagine living in a world where every time you outgrow your shoes you have to go back to the operating room,” Breuer said.

Angela would receive a biodegradable graft—a scaffolding made with bioengineered tissue seeded with cells from her bone marrow. It would connect the inferior vena cava—the vein that transports deoxygenated blood into the heart’s right atrium—with the arteries that carry blood to the lungs. She would require neither immune suppressants nor blood thinners. The bioengineered graft would also reduce the risk of infection. The operation would raise Angela’s oxygenation level, give her more energy, and lead to better growth and development.

“We wanted to choose the right patient for undergoing this procedure. Just as important, we wanted to find the right family,” Breuer said. “We talked to a number of people but felt the Irizarry family were quite a special family. They were probing and they asked appropriate questions.”

Building a blood vessel

Shinoka had used his technique in 25 patients in Japan. But when it came time to seek FDA approval, there were questions about how it worked. The FDA wanted to know what was happening during the graft’s formation and why. The answers would take years to find and fill 3,000 pages. (Support for the research came from the Yale Center for Clinical Investigation.)

Shinoka and Breuer believed at the time that bone marrow cells were a source of stem cells that turned into blood cells. But the bone marrow cells they were seeding onto the grafts disappeared after a week. They repeated the experiment, with the same results. If the stem cells were differentiating into blood cells, why did they vanish? After more experiments, which involved inserting a fluorescent dye into the cells so they could be tracked in mice, the researchers realized that even though they were getting the right result, they had misunderstood the mechanics of what was happening. The stem cells were not engineering new tissue. Rather, the stem cells were using molecules typically seen in inflammation to induce cells to leave nearby blood cells and regenerate. “It was much like how a salamander regrows its tail, or a starfish regrows an arm that’s been cut off,” Breuer said. “The scaffolding enabled the body to recreate a blood vessel. It enabled us to identify some of the molecular signals that were really important for this process, which we could then manipulate in our model systems.”

“It’s a milestone in tissue engineering,” said Gary S. Kopf, M.D., professor of surgery, who performed Angela’s surgery with Shinoka. “Dr. Breuer and Dr. Shinoka’s lab work is pioneering in terms of working out the mechanism of how tissue-engineered blood vessels form.”

“Moving this to the clinic is clearly a landmark accomplishment,” said Langer, the MIT professor. “This is a very significant achievement, that they have been able to take this very basic work in the laboratory and test it in people. You need to get the right biocompatibility. You need the right mechanical properties. You have to make it cell-compatible. You have to do it in such a way that there are no foreign body reactions.”

Between the lab and the operating room

The surgery began at 6 a.m. on a morning in August 2011, when the surgeons aspirated Angela’s bone marrow. They needed 5 ccs of bone marrow for each kilogram of her body weight, between 55 and 70 ccs total. The operating room team included two attending surgeons, one surgical fellow, two anesthesiologists, two perfusionists, and two nurses. A five-minute walk away in the Richard D. Frisbee iii Laboratory of Stem Cell Transplantation and Hematopoietic Graft Engineering, five postdoctoral fellows seeded the scaffolding with Angela’s bone marrow—a three-hour procedure. The timing had to be just right. The seeded scaffolding had to be ready when the surgeons needed it so as not to prolong the surgery. Before the operation the team practiced preparing the scaffolding more than 20 times with bone marrow purchased from a blood bank, Shinoka said. “We had stopwatches and we were trying to save every second we could,” Breuer said.

In addition, per FDA regulations, more than 10 tests had to be performed on the graft before it could be implanted to ensure that it was neither toxic nor infected.

While the fellows were preparing the graft, Shinoka and Kopf were in the operating room, preparing Angela’s heart. They needed about two hours to remove scar tissue from her previous surgeries. Throughout the operation the surgical team was in contact with the hematology fellows preparing the graft. “We were communicating every 30 minutes. The timing was pretty good,” Shinoka said.

Once the surgeons had the graft, which measures 18 mm in diameter by 4 to 5 centimeters in length, they connected Angela to a cardiopulmonary bypass machine for an hour while they implanted it. The entire operation lasted about eight hours.

The procedure, said Kopf, follows the path of the traditional Fontan procedure. “It is the same exact surgery,” he said, noting the one difference. “Instead of using a piece of plastic, usually Gore-Tex, we used the tissue-engineered graft. In terms of the surgery, I would say it is a little easier to use. It is thinner and more flexible and seems to hold the sutures very well. It doesn’t seem to have any bleeding, and the Gore-Tex does have a little more bleeding.”

Angela is not only the first patient in the United States to receive a bioengineered blood vessel but also the first of six patients participating in a five-year clinical trial. Breuer and Shinoka will monitor her for three years after her surgery. They will watch for complications from the graft and determine whether the graft does in fact grow with the child. Over time the surgeons plan to measure its size and compare it to normal blood vessels.

“We have taken a very cautious approach,” Breuer said. “We wanted to learn as much as we could from each patient before we went on to the next patient. We will do one patient, wait for six months; do two patients, wait six months; do three patients, wait six months.”

A child’s recovery

So far the surgeons have been pleased with Angela’s recovery, as have her parents. Angela has reached all her postoperative milestones, Breuer said. “The last time I saw her in clinic she was a normal 3-year-old running around. I think Angela will be a very normal child.”

“Before when she wanted to run with her brother, she got tired,” said Claudia Irizarry. “Now, she doesn’t want to stop. She can keep going. Her oxygen level is very, very good now.”

On a chilly day in December, Claudia kept her daughter inside, even though she’s strong enough to play outside and ride a bicycle. She has never attended day care or school, nor can she play with children other than her brother, because during her recovery she’s still susceptible to infections. At home, where she lives with her parents, brother, and grandmother, Angela likes to play games on the computer, watch Scooby-Doo on television, and curl up in her mother’s lap.

“If God sent me the baby, God also sent me the angels to care for her,” Claudia said. “I always believed that everything was going to be okay. She is very positive. She’s very happy. My hope for her is the same that all moms have, that she can do whatever she wants, that she can grow up and be a good person. I tell her I hope she grows up to be a doctor, to help kids the same way they helped her.”

Click here to see a video about Angela's story.