Researchers challenge centuries-old beliefs about limb regeneration

Ken Muneoka is no stranger to disrupting the field of regeneration; for example, in a 2019 ground-breaking publication in Nature, the Texas A&M University College of Veterinary Medicine & Biomedical Sciences (CVMBS) professor proved for the first time that joint regeneration in mammals was possible.

Now, his team is again challenging other centuries-old beliefs about the fundamental science of the field, this time related to how mammals might regenerate damaged parts of the body.

In humans, the natural ability to regenerate is limited to tissues like the epidermis, the outermost layer of skin, and some organs, such as the liver.

Other species, most notably salamanders, have the ability to regenerate complex structures such as bones, joints, and even entire limbs. As a result, scientists have been studying these species for more than 200 years to try to understand the mechanisms behind limb regeneration in the hopes of someday translating those mechanisms to induce more extensive regeneration in humans.

That research has led to a common belief that the single biggest key for limb regeneration is the presence of nerves.

While that may be true for salamanders and other species, it isn't the case in mammals, according to two of Muneoka's recently published studies. The first study, published last year in the Journal of Bone and Mineral Research, established that mechanical loading (the ability to apply force to or with an affected area) is a requirement for mammals. The second, published earlier this year in Developmental Biology, established that the absence of nerves does not inhibit regeneration.

Together, these findings present a sizeable shift in the thinking of how regeneration could work in human medicine.

What these two studies show counteracts the two-century-old dogma that you need nerves to regenerate. What replaces it in mammals is that you need mechanical loading, not nerves."

Ken Muneoka, Professor, Texas A&M University College of Veterinary Medicine & Biomedical Sciences

Importance of mechanical load

Scientists have long believed that two things must be present in an affected area in order to induce regeneration in mammals. The first is growth factors, which are molecules that can stimulate cells to regrow and reconstruct parts of the body.

In natural regeneration, these growth factors, which vary from species to species and by area being regenerated, are produced by the body. For human-induced regeneration, these growth factors must be introduced to the area.

The second factor believed to be necessary was nerves. This belief was predicated by many previous human-induced mammal regeneration studies on areas, usually digit tips, without nerves, in which the whole limbs were also no longer usable.

Those studies would have the predicted outcome – when growth factors were introduced regeneration did not take place–leading to the conclusion that, like in other species, nerves were a requirement for regeneration.

But the mechanical load aspect was ignored.

In their studies, Muneoka and colleagues decided to take a step back and ask the question, "is it really the nerves, or is lack of mechanical load part of the equation as well?"

Connor Dolan, a former graduate student in Muneoka's lab and first author on both new studies (who now works at the Walter Reed National Military Medical Center), came up with a way to test the denervation requirement in mammals that was inspired by astronauts.

The technique, called hindlimb suspension, has been used by NASA and other scientists for decades to test how mammals react to zero gravity environments. A similar process is used during medical procedures on legs of large animals to prevent the animals from putting weight on the affected limbs.

"Dolan found that when the limbs were suspended, even though they still had lots of nerves and could move around, they couldn't actually put pressure on their limbs so the digit tips wouldn't regenerate," Muneoka said. "It just completely inhibited regeneration."

As soon as the mechanical load returns, however, regeneration is rescued.

"Absolutely nothing happens during the suspension," Muneoka said. "But once the load returns, there will be a couple weeks of delay, but then they'll begin to regenerate."

That first step proved that even though nerves might be required, the mechanical loading was a critical component to regeneration.

Taking the research a step further, Dolan's second publication showed that nerves weren't required by demonstrating that if a mouse has no nerves in one of its digits but does in the others -; so that it's still exerting force on the denervated digit-; that digit will still regenerate.

"He found that they regenerate a little bit slower, but they regenerated perfectly normally," Muneoka said.

Ramifications of the research

Muneoka is quick to point out that their studies aren't saying that previous research is wrong, just that it doesn't directly apply to humans.

"There have been a number of studies in salamanders that prove that when you remove the nerves, they do not regenerate," Muneoka said. "Researchers have also been able to put growth factors they know are being produced by nerves into the cells and rescue regeneration.

"So, salamanders probably do need nerves to regenerate," he said. "But if we're going to regenerate limbs in humans, it's going to be a lot more like what happens in mice."

Since first beginning to look at regeneration more than 20 years ago, a number of Muneoka's ideas have pushed back against the generally accepted theories about regeneration. He said that getting these two papers published took almost three years because they originally tried to submit them together.

"Many scientists don't embrace this idea," he said. "A lot of people's careers are really dependent on their studies of nerves and how they affect regeneration. For a study to come out and say that for humans it's unlikely you'll need the nerves, the whole biomedical application of what people are doing in salamanders and fish kind of goes out the window."

Looking down the road

Nerves not being required for regeneration in mammals may seem like an academic point. After all, what would be the point of regenerating a limb if the person couldn't feel it or control it because it had no nerves. In that sense, nerves are still going to be an important part of the puzzle.

From Muneoka's perspective, the shift is that instead of thinking of nerves as a requirement for regeneration, nerves are a part of what needs to be regenerated.

Larry Suva, head of the CVMBS' Department of Veterinary Physiology & Pharmacology (VTPP), says the issue is that nobody was even thinking about the load aspect previously.

"Think of a blast injury where a soldier is left with a stump," Suva said. "No one, until this paper came out, was even thinking about a requirement from mechanical influences. You had people see that a denervated animal doesn't regenerate and they're thinking it's because the nerve was cut, but nobody was studying the mechanical load aspect."

As Suva puts it, science is full of people looking where the light is best.

"I work on bones, so when I see a problem, I look at the bone problem," he said. "People who work on nerves, all they look at are nerves. So it's very rare that someone like Dr. Muneoka will take a step back and take a more holistic view.

"That's what he brought to this idea, to this 200-year-old data," Suva said. "We now have to look at regeneration through a different lens because now we know the mechanical influences are extremely important."

One of the results of research focusing on nerves is that scientists have been able to recreate the growth factors that nerves produce, which has allowed researchers to start regeneration in salamanders, even if the nerves aren't present. Suva said that with these new findings, scientists will now know they have to do the same with the mechanical load aspect if they want to start regeneration in mammals.

"Scientists already have been able to trick the body into thinking nerves are still present," he said. "But now they know they'll also have to trick it into thinking there's a mechanical load, something that has not been done before."

Because cells react differently under mechanical load, somehow, that load is being translated biochemically inside the cell.

"There's a small number of labs looking at the biochemical basis for what mechanical load does to a cell," Muneoka said. "If we could understand that biochemical signal, then perhaps the physical force of mechanical load can be replaced by some sort of cocktail of molecules that will create the same signals in the cells."

The end of the road toward full human regeneration may still be a long way in the future, but Suva says that this kind of fundamental shift in thinking is a major marker on that road.

"Regeneration of a human limb may still be science fiction, but we know some facts about it, and now we know you have to have that mechanical load along with the growth factors," he said. "That changes how future scientists and engineers are going to solve this problem.

"There are still a number of complex problems to be solved before regenerating entire human limbs is possible, but Dr. Muneoka's findings are an important next step to make sure we're solving the right problems."

Source:
Journal reference:

Dolan, C.P., et al. (2022) Digit specific denervation does not inhibit mouse digit tip regeneration. Developmental Biology. doi.org/10.1016/j.ydbio.2022.03.007.

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