Modern Prometheus

Replacement organs printed with bio-ink will one day save thousands of lives. Sound improbable? Don't tell Gabor Forgacs. by Alan Bavley

Imagine being able to replace your diseased or worn-out organs with new, laboratory-fresh models made from your own cells. Your heart is failing? New muscle tissue or even an entire heart could be ordered up before a transplant becomes critical. Kidneys not working? Instead of dialysis, you could have a new one implanted. Such is the vision of Gabor Forgacs, the University of Missouri's George H. Vineyard Professor of Physics.

In Forgacs' basement laboratory, he is demonstrating that it is possible to "print" blood vessel-like structures. By methodically laying down circles of small spheres packed with human cells and then stacking layers of these circles one on top of another, he is able to create living cylinders of tissue, complete structures with the multiple layers of cells found in actual human veins and arteries.

Using the same techniques, he has taken cells from chicken hearts and built them into tissues that pulse just as the heart did. His goal is to make blood vessels that will be useful for surgery and, one day, build entire replacement organs in the laboratory.

Forgacs (pronounced FOR-gotch) is among the scores of researchers worldwide in the exciting field of tissue engineering, men and women who are pursuing the dream of building organs to order for the growing numbers of people desperately in need of organ transplants. Engineered bones, kidneys, hearts, and livers are all possibilities, each built from cells cultured from the individual patients receiving the organs. Tissue rejection would become a problem of the past.

Forgacs, who came to Missouri in 2000, brings a unique perspective to tissue engineering. Before working as a biologist, Forgacs started his scientific career in his native Hungary as a theoretical physicist. "Physicists are adventurous people," he says. "They want to go where the challenges are. Biology is an incredible challenge."

As a physicist, Forgacs expresses reservations about the time-consuming approach many biologists bring to tissue engineering. "Biologists are incredibly bright people. It's enlightening to see how they think about a problem. But I don't wish to spend a career solving problems by trial and error," Forgacs says. "We want to approach this practically."

Working with a $5 million National Science Foundation grant and a large international team of scientists, Forgacs has pioneered procedures for engineering tissues that differ radically from the conventional methods used by most other researchers in the field.

"He's a very creative force in science," said Stuart Newman, a developmental biologist at New York Medical College and member of Forgacs' research team. "Theoretical physics is as abstract and distant as you can get from the nuts and bolts of things, but he has a knack for understanding the practical side."

Classical tissue engineering employs scaffolds, structures in the shape of organs or tissues that are made of biologically friendly materials. These include natural ones such as collagen, or synthetics such as biodegradable polymers. The engineers deposit cells on the scaffold, keeping the cells nourished as they multiply and grow to fill the structure. If all goes well, the scaffold eventually biodegrades leaving a fully formed organ.

Anthony Atala at Wake Forest University has had the greatest success using this approach. He has built new, functioning urinary bladders. Seven children whose spina bifida left them vulnerable to incontinence and kidney damage already have the new organs. Further clinical trials are underway.

The big challenge to this approach, however, has been to find the right materials for building the scaffolds. Different cells need different environments. Complicating the search further, complex organs are made of a variety of cells. It took Atala years to discover the right formula for his engineered bladders, Forgacs says.

Not every researcher is so fortunate.

One researcher, for example, has been trying to grow blood vessels by seeding a cylindrical scaffold with cells. But the scaffolds have not biodegraded sufficiently to ensure that blood flows smoothly through the vessels. "The remnants of the scaffold still affect the structure of the vessels," Forgacs says.

This is where Forgacs and his colleagues innovate. His method for building blood vessels dispenses with scaffolds altogether. Instead, he takes advantage of new technologies and naturally occurring properties of cells that make the assembly of tissues look deceptively easy.

"People have been trying to engineer scaffolds with increasing complexity. You end up with something harder to reproduce," says Glenn Prestwich, an organic chemist at the University of Utah-Salt Lake City and another member of Forgacs' research team. "To be scaffold-free is really changing the paradigm."

At the heart of Forgacs' lab is a bioprinter, a custom-built machine about the size of a large refrigerator, that he is using to fabricate blood vessels. The bioprinter is decidedly not like the inkjet printers used with home computers, although other researchers have been modifying conventional inkjets for tissue engineering.

At Clemson University, for example, researchers have been able to print the university's unofficial tiger paw logo with bacteria. But that approach doesn't appeal to Forgacs.

"As fascinating as it is, there are problems," he says. With the heat, electricity and spraying, "an inkjet printer isn't a very friendly environment." And, in fact, a lot of the bacteria Clemson researchers used died in the printing process. Much of the published research about using inkjets has been to demonstrate that cells can survive the printing, Forgacs says, "that in principle it is possible."

The method also is very slow. Inkjets spit out single cells, but organs consist of millions of cells. So Forgacs uses a bioprinter that delivers millions of cells at a time. To make blood vessels, he cultures the three primary types of cells that make up the cylindrical organs: connective tissue cells called fibroblasts that comprise the outer layer, muscle cells that form the middle layer, and the endothelial cells that line the vessels. Then Forgacs combines the cells in the right proportions and forms them into tiny spheres, each about half a millimeter or less in diameter and containing 10,000 to 30,000 cells.

These spheres, Forgacs' "bio-ink," are packed into micropipettes and loaded into the bioprinter's printer head. Before printing starts, the printer lays down a gel film, the "bio-paper" that will accept the bio-ink. Once the gel sets, the printer pumps out individual spheres, making a circle about three millimeters in diameter. Then it lays down another sheet of bio-paper on top and prints another circle of spheres.

The whole process takes about 15 minutes. "We've managed to print six or seven layers at a time," Forgacs says. "But there is really no limit to it."

The stack of printed sheets matures in a "bioreactor," an incubator set to the right temperature and humidity and providing fluids pumped at the right pressure to mimic the environment of a blood vessel. In the bioreactor, nature begins to work its magic. Over the course of about a week, the individual spheres of cells slowly fuse together to form a solid tube.

But do these cells form something comparable to an organ? Forgacs found that indeed they do. When he printed the chicken heart cells, the beating of the cells was unsynchronized at first. But as the spheres melded, the cells began to beat in unison. This experiment demonstrated that, once these spheres of cells merge, they behave just like the tissue they came from.

Just as extraordinary, the cocktail of cell types in the spheres turns into the distinct tissues of a blood vessel. The fibroblasts migrate to the exterior of the cylinder, the endothelial cells travel to the interior walls, and the muscle cells find their way in between. "The cells know what they are supposed to do," Forgacs says. "This is nature. We just use what nature can do."

To build longer vessels, Forgacs has begun printing cylinders lengthwise, laying down parallel lines of cell spheres that widen and narrow as the printer goes up from one sheet to the next. Ever the physicist, Forgacs is engineering his blood vessels so that their physical properties are close to those of the real thing.

"We want to approach this practically," he says. "You might have the most beautiful tissue chemistry, but if it ruptures, it's no good. We want to build something a surgeon can use."

A competing method of building blood vessels is being used by Cytograft Tissue Engineering, a company in Novato, Calif., that has its vessels in clinical trials. In a Petri dish, they grow sheets of fibroblasts, the cells that form a vessel's outer layer. Then the sheets are rolled around a Teflon tube to shape them into cylinders. Endothelial cells are pumped into the newly created vessels. "The idea is ingenious," Forgacs says.

Recently, Forgacs and his colleagues made a further advance in fabricating blood vessels: printing vessels that branch out from each other as actual blood vessels do. "Nothing prevents us from branching," he says. "There's no method, other than ours at present, than can produce a branched tube."

Being able to create systems of branching blood vessels is a critical step towards the goal of fabricating new organs. It's not enough to grow tissues with the shape and function of organs; to survive, new organs must have the vasculature necessary to stay nourished with blood. "This is a serious issue [that] tissue engineering faces," Forgacs says. "It is our most challenging task. What I want to do before I die is to vascularize tissue."

The first use Forgacs sees for vascularized organ tissues is as a means for testing the toxicity of new drugs. Right now, safety testing is done with animals, but their physiology is an imperfect match with that of humans. Problems with some drugs, such as the heart attacks and strokes associated with the popular pain reliever Vioxx, may not be discovered until they've been on the market for years.

Forgacs sees the possibility of creating another step in the drug testing process. After trying a new drug in animals, the drug can be applied to living human tissues in the laboratory -- liver, kidney or, most likely, heart tissue. He is now working with a start-up company, Organovo, Inc. that would market these types of engineered organ tissue to pharmaceutical companies.

The biological questions confronting Forgacs today are very different from those that interested him after he received his doctorate in 1978 from Eotvos Lorand University in Budapest and the Landau Institute in Moscow.

"I was a brutally theoretical physicist. Nothing else much interested me than the calculations," he says. "Then you realize there are two-and-a-half people in the world who understand what you're doing, and they may not be interested."

In the 1980s, Forgacs traveled from campus to campus in the United States and Europe, avoiding the politics and impoverished laboratories of Hungary. Then, in 1990, while in the United States, a biologist friend invited Forgacs to his lab to consult. He became hooked on the subject.

Forgacs went back to school to learn biology at Clarkson University in Potsdam, N.Y., the Woods Hole Marine Biological Laboratory in Massachusetts and finally, to Princeton University. When he was beginning this new career, it was fairly uncommon for physicists and biologists to talk much to each other. He recalls having trouble engaging other biologists at Princeton.

"They'd say something like, 'You know, physicists are really smart people, but I've got to go now,'" Forgacs says. "I had to convince people that I knew about biology. It's common now, but then I was an oddball."

"Nowadays, physicists and biologists are talking to each other. They should. But there is still a vocabulary gap."

Forgacs has tried to help bridge that gap by co-authoring, with Stuart Newman, Biological Physics of the Developing Embryo, a textbook that discusses physical principles essential to biology. "I take the attitude, 'Why separate those things?' That's an artificial separation," he says.

Meanwhile, Forgacs sees more physicists like himself flocking to biology. One reason is financial. Federal funding for the life sciences skyrocketed in the 1990s, outpacing growth in the physical sciences, he says.

Another reason is intellectual. Biology has progressed to the point where it is asking questions that physicists find irresistible. "Biology is made up of self-organizing systems that have so many variables, so many feedback systems," he said. "It's just damn complex."

While working 18-hour days, Forgacs still makes room for other aspects of his life. Every 10 days he travels to Potsdam, N.Y., where his wife, Marta, a pediatrician, still maintains her practice.

While in New York he's completed the New York City Marathon five times, including a race that he ran with his son, Andras, who lives in the city. While not quite in marathon condition right now, Forgacs may try running the marathon a sixth time. After all, he says, physicists love challenges.

Man and Machine Gabor Forgacs with his bioprinter, a custom-built machine about the size of a large refrigerator. Forgacs is using the bioprinter to engineer branching blood vessels, a critical step toward the goal of fabricating new human organs.