The graft that keeps on giving
by Nancy Fliesler
Researcher Dario Fauza is determined to
create living patches to close congenital holes in everything from tracheas to diaphragms.
When Dario Fauza, MD, came to Children's
Hospital Boston from Brazil in 1992 as a young pediatric surgeon, his sole purpose was to hone his surgical skills and learn the use of extracorporeal
membrane oxygenation (ECMO), a form of heart-lung bypass for children with severe cardiac or respiratory problems. He fully intended to return home when his
fellowship ended. But in 1995, Fauza cared for a baby who changed the course of his career.
The baby boy had a completely normal, beating heart, but due to a large opening in the chest wall, the heart was outside his body. "It was so far out of his chest that it was kinking his blood vessels, so he needed ECMO," Fauza recalls. He and his colleagues tried to cover the exposed heart using whatever bits of skin they could borrow from elsewhere on the newborn's tiny body, but it just wasn't enough.
Frustrated, Fauza felt that if a large enough "patch" of the right material were available to close up the chest, the baby could have been saved. Perhaps a patch could have been made to order while the baby was still in the womb by taking a few of his cells and growing them into muscle, cartilage and skin tissues for use after birth. Or perhaps his chest defect could have been repaired in utero, before it became a serious problem.
Fauza took a second fellowship at Children's to work on these ideas, and today has his own research lab, which works closely with the hospital's Advanced Fetal Care Center (AFCC). It's unlikely he'll move back to Brazil anytime soon.
When a baby is born with a structural defect, surgeons have to be creative in finding a fix. Time is often of the essence, and newborns may need to be sustained on ECMO or artificial ventilation while they await operations. Surgeons often try grafting in tissue from elsewhere in the body, but this can cause complications, and newborns may have too little of the right kind of tissue to work with, especially if born prematurely. Patches made of synthetic material like Teflon are used for some repairs, but they don't work well in growing children. And if finding an immunologically matched adult donor organ is difficult, finding one for an infant is close to impossible.
So Fauza began investigating the idea of growing new tissues and organs for these tiny patients, using their own fetal cells so there would be no risk of the immune system rejecting the grafts. And since fetal cells are immature and not fully specialized, they could be used to generate a variety of tissues.
Perhaps the best attribute of fetal cells is that they can be easily obtained from amniotic fluid, which bathes the fetus in the womb. Millions of pregnant women elect to have amniotic fluid drawn in the 16th week of pregnancy to test for chromosome defects, the procedure known as amniocentesis. And when a prenatal ultrasound reveals fetal malformations, amniocentesis is done routinely.
Despite these important advantages, tissue engineers have generally fash-ioned tissues using adult cells.
"Tissue engineering has been around since the 1980s, but surprisingly, no one had thought of using fetal cells to treat a birth defect," says Fauza. "Fetal cells are the best cells you can have. They grow very well, and they're very plastic—you can coach them to do what you want."
In his initial animal studies, Fauza fashioned tissues using cells snipped out of the mother's placenta, or from the fetus itself, but extracting the cells from the fetus or placenta poses a risk of pregnancy complications. In amniocentesis, the fetus and placenta aren't touched at all: fluid is drawn out via a needle inserted into the mother's uterus through her abdomen. Complications are rare.
"In many cases, the amniotic fluid is collected anyway," says Fauza. "It's a precious resource that's eventually thrown out, but shouldn't be."
Many types of cells float in amniotic fluid, but Fauza is focusing on so-called mesenchymal stem cells for most of his repairs. "Mesenchymal cells are abundant and easy to isolate, and they give rise to many of the tissues surgeons need," he says.
These valuable fetal cells have about one-third the creative potential of embryonic stem cells, from which they're directly descended. While embryonic stem cells can generate all tissues in the body, mesenchymal stem cells specialize in making connective tissues, including muscle, bone, cartilage, fat and tendon-forming fibroblasts.
Whether these cells come directly from the fetus or the placenta is a
matter of debate, but less than two tablespoons of amniotic fluid should provide enough of them to repair a human malformation in utero after birth, or, potentially, years later.
Fauza's team multiplies the cells in culture and "seeds" them onto a customized, biodegradable scaffold that helps the cells organize themselves into a tissue of the needed dimensions and shape. The tissue can then be manipulated—by exposing it to an electrical current, magnetic fields, mechanical stress, or genetic or chemical factors—to give it the desired biophysical properties. Once the engineered tissue is implanted in the body, the scaffold gradually dissolves, leaving just the living tissue.
Among Fauza's first surgical applications was repair of congenital diaphragmatic hernia, or CDH, a birth defect in which the diaphragm—the membrane that separates the lungs from the visceral organs—has a hole in it. If the hole is large enough, the stomach, intestines, spleen, and liver can end up in the chest cavity, crowding the lungs and preventing them from growing normally. The result is serious respiratory distress for the baby.
CDH is one of the most common major birth defects—affecting about one in 2,800 live birthsóand isn't well addressed by present techniques. Currently, surgeons close up large holes in the diaphragm by suturing in a patch made of Teflon. However, the patch eventually tears loose in more than 50 percent of children because it doesn't grow along with the child, requiring one or more operations to mend tears. (At Children's, a referral center for CDH cases, the tear rate is around 35 percent.)
Last year, Fauza reported successful CDH repair in animal studies, using a patch made of tendon engineered from mesenchymal cells. His report, pub-lished in the Journal of Pediatric Surgery in June 2004, marked the first use of a tissue-engineered graft derived from amniotic fluid cells in a live animal.
Fauza's team isolated the mesenchymal cells, allowed them to proliferate and added about 100 million to a gelatinous solution made of collagen. They poured the solution into a Petri dish and allowed it to firm up, forming a roughly credit-card-sized patch. On the day of surgery, the team took the patch out of incubation and assembled the actual "construct" for implantation, adding a supportive base made of a leathery material and wrapping it in a porous membrane designed to hold the elements together while allowing blood vessels to penetrate. To provide an initial blood supply, the construct was topped off with omentum, a fatty tissue rich in blood vessels.
A year after the repair, the animals' diaphragms showed good healing: the tendon grafts had grown with them, had a good blood supply, were well integrated into the surrounding tissue and showed good tensile strength on mechanical testing.
Fauza now hopes to conduct what would be the first clinical trial of fetal tissue engineering to repair a birth defect in a child. If approved by the FDA, he and surgical colleagues Jay Wilson, MD, and Russell Jennings, MD, will enroll 20 pregnant mothers with a prenatal ultrasound diagnosis of CDH. Amniotic fluid would be collected several months before birth and a tissue engineered patch made ready for use soon after delivery.
Because the patches would be made of the babies' own cells and a natural scaffold already approved by the FDA, Fauza's team could skip the Phase I safety trial and go directly to Phase II.
Tissue-engineered sections of trachea may be next in the pipeline. Congenital tracheal defects range from complete absence of the trachea to incomplete or malformed tracheas, but they're all life-threatening: babies born with defective tracheas cannot breathe and must immediately go on ECMO, which can cause complications like neurologic deficits.
Surgeons have tried an array of approaches, with mixed results: grafting in pieces of the baby's rib or pelvic bone; implanting synthetic substances; placing a stent in the hope that the tissue will scar around it and form a tube; or simply suturing the ends together if enough tracheal tissue is present. "These are all makeshift solutions," says Fauza. "They're fraught with complications—infection, reoperation, narrowing of the trachea."
Unlike CDH, which can be repaired after birth, Fauza is advocating for fixing the trachea while the baby is still in the womb. Tracheal surgery in a newborn requires that the baby be intubated and ventilated for a long period after the operation to allow the trachea to heal.
Fetal surgery would eliminate these interventions and the complications they can cause. "The fetus doesn't need the trachea, so the repair would have time to heal in utero," Fauza explains. "And fetal healing is very good—in fact, for reasons not yet fully understood, it's better than adult healing."
Fauza has already fixed tracheal defects in animals using tube-shaped grafts (see photo) grown from cartilage cells taken from the ear. All five animals were able to breathe spontaneously at birth. Now his lab is crafting cartilage tubes from the amniotic fluid, using a special culture medium to induce the mesenchymal stem cells to specialize as cartilage cells.
Also in the pipeline are tissue-engineered grafts to fix structural cardiac anomalies, the most common birth defects. Currently, surgical treatment is limited to heart transplantation or augmentation with prosthetic patches, which often fail or cause complications. In a recent animal study, Fauza's team used cells from fetal skeletal muscle to engineer heart grafts. Once implanted, they began to take on the characteristics of heart-muscle cells—an important finding since it eliminates the need to biopsy the fetal heart to get cells. Fauza is now working on deriving heart cells from amniotic fluid.
And last year, he reported early results implanting neural stem cells into spinal cords damaged by spina bifida, the most common permanently disabling birth defect, in which the spinal column doesn't close fully during development. The exposed cord is highly vulnerable to damage, saddling children with a variety of problems, including hydrocephalus (fluid on the brain), full or partial paralysis, bladder and bowel incontinence, urinary infections and learning disabilities.
A large multicenter trial is being held to test surgical closure of the spine in utero, but it has been shown that while closure prevents future damage, it doesn't reverse existing damage. "Instead of just closing the defect, why don't we put in some neural stem cells?" Fauza reasoned. In preliminary studies, he injected neural stem cells into the spinal cord and later found that neural growth factors were being produced in the cord around the injection site. Fauza's team is now working on a
tissue-engineered construct bearing neural cells that would be wrapped around the cord. Fauza also speculates that the cells could eventually be obtained from amniotic fluid, because in patients with spina bifida, cerebrospinal fluid leaks out of the spinal column, carrying neural cells with it.
Fauza envisions a future in which amniotic fluid is banked for everyone's use. His lab is conducting studies to see if amniotic-fluid cells could be used by people other than the donor without causing an immune reaction. "Fetal cells are less immunogenic than adult cells, and amniotic-fluid cells are even less immunogenic than fetal cells," Fauza says. "This is just the beginning; the dawn of a new avenue of treating birth defects. We're excited to be
pioneering it."
To learn more about supporting tissue
engineering research at
Children's Hospital Boston, contact Lynn Susman
in the
Children's Hospital Trust at (617) 355-5344 or lynn.susman@chtrust.org.
