“War is cruelty,” said General William Sherman, and one measure of the cruelty suffered by today’s soldiers in Iraq is the number of amputated limbs. Radical improvements in emergency triage, medical evacuation and body armor mean more soldiers are surviving battlefield trauma than ever before. The unfortunate corollary is that more survivors are living with harsh injuries.

In World Wars I and II and the Korean War, fewer than 2 percent of total casualties were amputations. The wars in Afghanistan and Iraq, however, have produced amputations at more than twice this rate. As of June 1, according to the U.S. Army Medical Command, 607 American veterans of Afghanistan and Iraq were without one or more limbs. But two new military-sponsored research projects in regeneration could revolutionize an amputee’s prospects.

Here’s the futuristic vision: Were a soldier’s eye tissue to be scorched by chlorine gas, his intestine ruptured by small arms fire, or her arm blown off by a roadside bomb, such a soldier might be transported to a regenerative medicine complex where, ideally, his or her own cells would be put to work to create new ocular tissue, new intestinal tissue or a new arm. This vision applies easily to non-military needs as well—the diabetic whose limb is amputated, the machinist who accidentally loses a finger, the infant born without arms, the elderly person with macular degeneration.

A project funded by the U.S. Department of Defense seeks to heal a wound’s damaged tissue by regenerating the tissue as opposed to the body’s routine healing method of creating scar tissue. The project is run by the Department’s chief research and development agency, the Defense Advanced Research Projects Agency, or DARPA, which has a record of pursuing high-risk, high-payoff science. Last year, it launched Phase 1 of the Restorative Injury Repair Program, or RIR, which has received $14.4 million in funding so far, according to spokeswoman Jan Walker. Injuries that might be healed regeneratively range from penetrating wounds to “chemical and thermal burns, musculoskeletal injuries, blast overpressure, etc.,” according to DARPA.

A smaller but equally innovative initiative called the Soldier Treatment and Regeneration Consortium, or STRaC, is already heading toward preclinical and clinical studies. STRaC’s partnership of military, academic and industry researchers, which include the U.S. Army Institute of Surgical Research, the U.S. Army Medical Research and Materiel Command, the Walter Reed Army Medical Center, the Wake Forest Institute for Regenerative Medicine, the Trauma Institute of San Antonio and Honolulu-based company Tissue Genesis, have been given the task of growing a fully functioning finger by the year 2011.

For the RIR program, the first benchmark is even more immediate—and more daunting. By the end of Phase 1—or May 2008—researchers must get a blastema to form in a non-regenerating wound site in a mammal, or risk losing their funding. A blastema is a smallish mound of cells that appears at the margin of, for instance, a lobster’s lost claw, from which sprouts a new claw. When a lobster drops its claw, the wound sends out molecular signals which inform nearby cells to dedifferentiate; that is, they become less specialized and revert back to a stem-cell-like state. These cells, which form the blastema, in turn yield the wide range of specialized cells that are needed to remake the limb and all of its various tissues.

Wounded animals that create blastemas generate new tissue rather than scar tissue. Therefore, the blastema represents a pivotal biological entity—a boundary, as it were, between scarring and regeneration that scientists hope to better understand. The immediate hitch is that mammals don’t normally produce a blastema, at least not the kind that results in the recreation of a full-fledged appendage.

“DARPA doesn’t go for baby steps,” says Susan Braunhut, a professor of biological sciences at the University of Massachusetts at Lowell and an RIR participant. “They have asked us to do what has never been done before. This is not incremental science. They believe, and I believe along with them, that we are at a point in science where this has been made possible.”

With no laboratories of its own, DARPA has assigned the project to two research teams that together represent an interdisciplinary mix of scientists from nine academic research labs and one biotech company. One team is coordinated by Stephen Badylak, the director of the Center for Pre-clinical Tissue Engineering at the University of Pittsburgh’s McGowan Institute for Regenerative Medicine. Ken Muneoka, a Tulane University molecular biologist who specializes in vertebrate limb formation, heads the other group. The program is slated for two phases, each of which will last two years.

The RIR program and its strikingly difficult assignment might not have made it onto DARPA’s agenda were it not for several recent developments. First, it was only in the mid- to late-1990s that researchers on several continents arrived at a basic understanding of what happens in the blastema at the cellular and molecular levels that enables an animal to grow back a missing appendage, be it a lobster’s claw, a newt’s leg, or a snail’s mantle. While the process of cells dedifferentiating back into a stem-cell-like state so that prodigy cells can then re-specialize to form tissues anew is a vital part of producing a new limb, it’s not the whole story, says Jeremy Brockes, a biochemist at University College London. Brockes, who has been at the forefront of this research for many years, notes that cells from surrounding tissues, like the dermis of the skin, influence the creation of the new limb but don’t necessarily dedifferentiate to do so.

A second area where scientists are making progress is the study of gene expression and the ability to identify genes that either promote regeneration or block it. A third advance has to do with signaling molecules, about which, the McGowan Institute’s Badylak says, “we know one hundred times more today than we did five years ago.” The extracellular matrix that exists between cells is chockfull of proteins and smaller signaling molecules that play an integral role in regulating regeneration. Since some of these signalers encourage tissue to regenerate, rather than to scar, identifying such factors is a priority for RIR scientists.

“The idea here,” says David Gardiner, a developmental biologist at the University of California, Irvine, who is also participating in RIR, “is that regeneration is a basic biological property and widespread in animals.”

Indeed, the so-called diva of regeneration, the salamander, can regenerate its fore- and hind limbs, tail, spinal cord, optic nerve, retina and lens, a section of the heart’s ventricle, and its upper and lower jaw, while a mammal can do none of these things. Still, a mammal is far from a regenerative laggard.

Take a human fetus, whose appendages retain a high degree of regenerative ability up through the 16th week of gestation. And a healthy human adult, who loses an estimated 10 billion cells each day—from intestine, skin, hair, blood, bone, muscle and elsewhere—renews them in good time. Our livers can grow back after as much as one-half is removed. Fingertips in both children and adults can also regenerate. “Sure,” the DARPA quest is incredibly difficult, allows Jon Mogford, RIR program manager, “but we aren’t asking the human body or a mammal to do something it’s not capable of doing.”

Although “the seedling” for the RIR program was planted a year or two before 9/11, when the Iraq war came along, there was a shift in the federal allocation of research funds, says David Vorp, associate professor of surgery and bioengineering at the University of Pittsburgh. “Research dollars earmarked for biomedical and clinical applications decreased, and funds increased for defense research having to do with soldier fixing and healing” he notes. “The funding trend is away from more traditional applications to beginning to think outside the box.”

But questions remain: Is DARPA’s goal of replacing scar tissue with regenerated tissue too ambitious? Will it be possible to talk the adult body out of its normal response to a wound—healing through scarring—and talk it into developing new tissue the way it did as a fetus? Once an injury occurs, scarring immediately commences—can it be immediately halted?

Brockes, who is not participating in RIR, agrees with the project’s objective of attempting to derive a mammalian equivalent of a blastema. “If I were going to work on trying to get human limbs to regenerate, I would choose that goal as well,” says Brockes, who discussed this very possibility—engineering a blastema—and the prospects for limb regeneration in adult vertebrates in a 2005 Science article. What makes this a particularly reasonable approach, he adds, is that the blastema is an autonomous structure.

Experiments from the 1970s demonstrated that when a salamander blastema’s mound of cells is transplanted from the margin of a limb stump to, say, the fin or even the chamber of the eye, “it will go ahead and give you a leg,” Brockes says. This is due to blastemal cells having what is called positional memory. In a salamander, cells at the margin of the loss know unequivocally that they are going to create a leg, and nothing can change their mind. The instructions for “leg” are encoded in their DNA.

Many tissue engineers might think that a better approach to replacing a leg than prompting a blastema to form would be, in the tradition of tissue engineering, to seed a three-dimensional scaffold made of dissolvable plastic with cells, extend the scaffold out from the limb stump and wait for the scaffold’s cells to mature into the desired tissue and structure. “But the fact is, nature doesn’t do this,” Brockes says. “Nature recreates at whatever level you amputate the limb.” Tissue engineering a new limb, Brockes believes, would be tremendously difficult, for it would hinge on figuring out complex interactions between molecules and different cell types so that the leg’s necessary tissues—bone, muscle, blood and skin—would align properly. It appears more promising to get cells in the blastema to do the work for you.

Brockes, who, on the whole, believes that humans might someday regenerate their appendages, cites another reason for optimism. In nature, he notes, even closely related species vary in their regenerative prowess. The only salamander species that can regenerate the lens of its eye, for instance, is the aquatic newt, whereas an axolotl, although a fabulous regenerator of other structures, lacks the ability to regrow its lens. Such examples add weight to the idea that the potential for regeneration dwells in all species, that those species that can’t regenerate an entire structure have lost some biological component that would allow them to do so, and that therefore, with some prodding, even humans might recover the capacity.

Muneoka, the Tulane molecular biologist, who has been studying how limbs regenerate for 28 years, has two additional reasons for believing that humans might be cajoled into re-growing complex parts. Years ago, he recalls, when he began studying salamander regeneration, “I asked myself, how different is limb formation during [in utero] development from the same process in regeneration” in an adult animal. “The answer is, they are very similar. I realized that if that’s the case, and humans grow limbs during development, why shouldn’t they be able to regenerate a limb?”

His second reason is based on what he knows about fibroblasts, the cells that in humans produce the collagen fibers in scar tissue. It turns out that in salamanders, fibroblasts, instead of leading to scarring, trigger the regeneration response. “Fibroblasts in both humans and salamanders carry spatial information critical for identifying what parts of the body are missing,” Muneoka notes.

Researchers have found that if an animal scars, it doesn’t regenerate; and if it regenerates, it doesn’t scar. The implication of this antithetical relationship is that if you could close the door on scarring in humans, you might open it to regeneration. “That’s certainly one step, but it probably wouldn’t be enough,” says Muneoka. He and his RIR teammates, therefore, while training their sights on fibroblasts, are also investigating other early events that occur after an injury—the inflammatory response, for instance—to gain new insights into how the scarring process might be redirected.

Badylak’s team, meanwhile, is studying the extracellular matrix. “We’re asking the question, ‘How can we harness some of the biological signals in the extracellular matrix to change the way an adult mammal heals?’” he says.

Badylak, who is an unusual combination of M.D., Ph.D., and veterinarian, stumbled across the extracellular matrix’s extraordinary capacity for regeneration more than 20 years ago. He happened to extract a layer of intestinal tissue in a dog that, when transplanted, yielded functioning tissue for the dog’s aorta.

What he has discovered since is that matrix harvested from the right place, when applied to a wound, invites regeneration and discourages scarring. The procedure can be used for reconstructing the urinary bladder, treating a torn rotator cuff or diabetic foot ulcer, replacing the lining of the brain (dura mater), and repairing a large or small finger cut. A powder version of matrix harvested from pig intestine reportedly was responsible for growing back three-eighths of the outer digit of a man’s middle finger—bone, nail and all.

As for smaller cuts and burns, “people in the lab swear by it,” says Scott Johnson, a staff scientist in Badylak’s lab. The reason the matrix does not elicit an immune-system response, according to Badylak, is because it consists mostly of proteins and other highly conserved molecules, and contains no cells. Findings connected to extracellular matrix have spawned some 40 patents and have also resulted in clinical treatments from a unit of Johnson & Johnson and the biotech enterprise Cook Group of Bloomington, Indiana.

Badylak’s RIR team includes a tissue engineer, two cell biologists, an immunologist, a pharmacologist, and a specialist in amphibians, each of whom is coming at the single objective of minimizing scarring and maximizing regeneration from a different angle:

• Badylak’s McGowan lab is studying biological signals within the extracellular matrix;

• Braunhut of the University of Massachusetts is running experiments to see whether injected matrix can prompt growth in the toes of mice;

• Shannon Odelberg at the University of Utah is working to identify genes that make the extracellular environment appropriate for regeneration and, therefore, for the blastema’s formation;

• Ellen Heber-Katz at The Wistar Institute is looking at the role of immune cells and the inflammatory response;

• Lorraine Gudas at Cornell University is studying cell differentiation;

• Hans-Georg at Northwestern University is testing the ability of regulatory genes to control the generation of stem-like blastema progenitor cells from muscle.

These teammates are happy to be working with people outside their disciplinary silos. “Here I was in wound healing, and I’ve never talked to a salamander regeneration person,” says Braunhut. “Why wasn’t I talking to them?”

Meanwhile, the matrix powder, which Badylak sees as strong evidence that mammals can be directed to regenerate without scarring, is the centerpiece of a clinical trial connected to the Defense Department’s STRaC initiative. Researchers at the U.S. Army Institute of Surgical Research in Fort Sam Houston, Texas, are testing the effectiveness of extracellular matrix by applying it to the finger stumps of five soldiers who have recently lost all or part of their fingers. The powder is to be administered for two weeks, and then the fingers’ length, function and sensitivity will be measured for several months.

“The idea here was to take some of Steve’s technology straight to soldiers,” says Alan Russell, director of the McGowan Institute. “No one thinks we’ll go past a joint. But even if you could grow back half a centimeter, you would still give a person a better quality of life.”

Investigators are both guarded about their experimental mission—they admit that getting a blastema to form in a mammal by next May is a mighty tall order—and determinedly positive. “All you have to do is look at history, and you know these things can happen,” Badylak says. “Can you imagine if you said, in the year 1900, that we would be able to transplant hearts? You would have been laughed out of town. And now look. Here at the University of Pittsburgh, we transplant an organ every 18 hours.” 

Ann Parson is the author of The Proteus Effect; Stem Cells and Their Promise for Medicine, which was chosen by Library Journal for its yearly list of best science books for general readers.