The injured soldier, a patient at Walter Reed Army Medical Center in Washington DC, was unforgettable. “He was a special forces guy, in the middle of a firefight,” his physician Leon Nesti recalls. “His leg was blown off above the knee. In the middle of this firefight, he picked up his leg and used it to stabilize his weapon, and kept firing at the enemy.”

When the soldier arrived at Walter Reed about 4 years ago, Nesti says they had to take him to the operating room every couple of days to clean the wounds and remove damaged muscle tissue. Finally, he was fitted for a prosthetic.

But about a month later, his prosthetic became painful. The soft tissue that attached to the device had turned hard and stiff. He had developed a disorder known as heterotopic ossification, where bone begins to grow in areas where there should be muscle.

It was a condition Nesti had been seeing frequently in injured soldiers from the wars in Iraq (where this soldier had been based) and Afghanistan, and he wondered what was happening in their bodies. The disorder occurs in the civilian population as well, sometimes in victims of car crashes and even some athletic injuries, but its etiology and mechanism are poorly understood. And, it was occurring in these soldiers at rates previously unheard of—as many as 65 percent of amputees were developing heterotopic ossification. Previous research has documented heterotopic ossification in only one-quarter of patients who had abdominal surgery or hip replacement. (J Comput Assist Tomogr., 32:872–76, 2008; J Arthroplasty, 2009 e-pub June 2.)

Nesti wondered if there was a clue in the discarded tissue surrounding the injury that could shed light on why their tissue ossified so frequently. To take a closer look at the soldiers’ tissue, Nesti contacted Rocky Tuan, now the director of the center for cellular and molecular engineering at the University of Pittsburgh, and Nesti’s postdoc supervisor.

Along with other members of Tuan’s lab at the National Institutes for Health, they analyzed samples of the tissue extracted from the wounded soldiers during surgery. What they found shocked them. The traumatized muscle tissue was chock full of stem cells—nearly 4,000 times the density found in bone marrow and considerably higher than what has ever been found in normal muscle tissue (J Bone Joint Surg Am., 90:2390–98, 2008).

Further analysis revealed that the stem cells were already being pushed to specify into a certain cell type, expressing more osteogenic and nerve-generating factors. (J Orthop Res. 2009 Jun 10, e-pub ahead of print).

Tuan and Nesti think that the proliferation of progenitor cells following a serious injury is an overzealous healing response, which leads to heterotopic ossification. “It makes sense, that if the body is injured it sends off signals to recruit stem cells to the site of the injury to speed up the healing process,” says Nesti. But, why these cells seem to be differentiating into bone as opposed to muscle is still a mystery.

George Muschler, a biomedical engineer at the Cleveland Clinic, agrees that the soldiers’ heterotopic bone formation likely traces back to these progenitor cells, perhaps to issues during their recruitment to the site of the injury and differentiation. These excess progenitor cells are also probably a “central element in wound repair and tissue regeneration,” he adds.

Robert Pignolo, director of the Ralston-Penn Clinic for Osteoporosis and Related Bone Disorders in Philadelphia, also agrees that the cells are likely behind the bone formation, but cautions that stem cells often behave differently in culture than live tissue—so just because they exhibit osteogenic properties in vitro, does not mean that they will form bone when implanted in a living organism.

Nesti and Tuan are also evaluating the progenitor cells’ therapeutic potential, particularly for peripheral nerve regeneration (J Tissue Eng Regen Med., 3:129–38, 2009). The cells secrete nerve-inducing factors, says Tuan, and recent unpublished experiments by him and Nesti have suggested that they can speed up the process of nerve regeneration.

The fact that much of this research was made possible because of the wars is not lost on either Tuan or Nesti. “It’s one of the few positive outcomes of war, I guess,” says Tuan.

Gary Brooke writes:"Henceforth, either due to prior osteo-lineage commitment or due to stimuli such as pressure, these cells differentiate down the osteocyte lineage and subsequently produce bone tissue."
Gravitational pressure is "dead" pressure, and sound pressure is "alive". Cells develop in a vibrating environment, VAT is using long time (several hours below 75 dB) sound exposure and may have a different effect from stump/prothesis pressure. This phenomenon ought to be tested out.

I have been working with Vibroacoustic Therapy (VAT) for more than 30 years, and we have more than 40 000 hours of evidence based experiences. No statistical data to speak of. We have seen strange effects on brain stroke patients whi regain some movement after several years of no progress. Also post operative scarring feels to be softened both shortly after operation and after years of discomfort. I wonder if the phonons in whole body vibrations can alter the function of cells inter- and intracommuncation behavior to a degree that the described ossification process could be changed in a positive direction so that continuous 40 Hz (?) vibration might alter the behaviour of the stem cells?
This just a long distance shot from
Olav Skille

I would suggest that due to the severe nature of the trauma, sufficient mesenchymal stem cells have migrated out of injured bone and have engrafted inappropriately in muscle tissue. As these are stem/progenitor cells, they can hang around for some time and expand in number. Henceforth, either due to prior osteo-lineage commitment or due to stimuli such as pressure, these cells differentiate down the osteocyte lineage and subsequently produce bone tissue.

Thanks for your question. The answer as to why soldiers' tissue ossified so frequently is here: Tuan and Nesti think that the proliferation of progenitor cells following a serious injury is an overzealous healing response, which leads to heterotopic ossification.

However, why these injuries lead to ossification more often than other injuries is, as yet, a mystery.

Alison McCook
Deputy Editor

Did I miss the answer or misunderstand the question? Why was this occuring more frequently in these injured soldiers?

Could the pressure effect of the prosthesis be leading to the ossification? Would delaying the placement of prostheses help in reducing that process? What about the tightness of the skin in the stump itself?

As stem cells are stem cells, and with such massive numbers, are there some major regenerative processes that could taken advantage of? For instance, could an entire section of limb be re-created over time? All of those stem cells might be able to do something substantial if a tight stump were not made but the amputated limb were allowed to expand, possibly with the application of electrical fields.