Every week, Michael Levin receives calls and letters from people who have lost limbs.
How this happened varies — industrial accidents, military injuries, birth defects, the list goes on — but they approach Levin, a professor and director of the Center for Regenerative and Developmental Biology at Tufts University, with the same question: When can they regrow their limb?
Scientists project that by 2050, approximately 3.6 million Americans will live with the loss of a limb. While technologies like prosthetics have advanced, doctors are still unable to induce human limb regeneration.
But scientists are a step closer. In a study published Wednesday in the journal Science Advances, Levin and his colleagues announced they were able to trigger the regrowth of legs in adult frogs.
“They weren’t perfect cosmetically, but they were pretty darn good legs,” he said.
The regrowth was facilitated by a combination of a five-drug cocktail and a silicone wearable bioreactor called a BioDome. The cocktail was developed by Levin and the study’s first author Nirosha Murugan, now an assistant professor at Algoma University in Ontario, Canada, and designed to to contain ingredients that spurred different actions, including the inhibiting of collagen production, which causes scarring, the encouragement of nerve fiber, blood vessel and muscle growth, and the tamping down of inflammation.
This cocktail was also the first the team tried — the initial stab at getting the right combination of ingredients and doses.
“This makes me very happy because if this is our first guess and it’s this good, imagine what the optimized version is going to look like in the future,” Levin said.
In the experiment, 115 female African clawed frogs were assigned to one of three treatment conditions after having a hind leg amputated: the BioDome with the cocktail treatment, just the BioDome, or a control experience of no intervention. As tadpoles, these types of frogs can regenerate legs but lose the ability to do so when they are adults.
The BioDiome was used within the first 24 hours of amputation — and in the case of one group of frogs, was loaded with the five pro-regenerative compounds. This brief treatment activated an 18-month period of regrowth, resulting in almost fully functional legs. The new limbs contained bones, muscle and nerves. The frogs could use them to stand and to swim.
The legs didn’t have the same webbing and long toes these frogs typically have. However, Levin doesn’t know if that’s because the team hasn’t figured out the right cocktail yet, or because they didn’t wait long enough for them to develop.
There was also some variability. “Some animals responded great, and some animals not as great as others, but everybody got better,” he said.
Even the BioDome with no drugs had a useful effect for some frogs, facilitating partial limb regrowth likely because it still provided a protected environment. While animals such as flatworms, starfish and crabs regenerate limbs, mammal injury tends to result in scar tissue, which prevents blood loss and infection. This enables survival and is, in part, a result of injuries being exposed to air — but it may also explain why humans don’t grow arms back like a salamander can do.
A hydrated, protected environment, like a pond without predators or, in this case, a BioDome, allowed the frogs’ bodies to put their efforts toward regenerating, Levin said.
Levin’s lab researches how cells make decisions and how to use this process to kick-start regeneration. Other approaches in regenerative medicine focus more on tissue engineering and the actual construction of new body parts. Levin subscribes to a different school of thought which approaches regenerative medicine by taking advantage of the capabilities of cells and rousing them into action. He compares it to focusing on software rather than creating hardware.
“Cells already know how to make all the organs in your body — they did it once during embryonic development and that information hasn’t gone anywhere, it’s still there,” he said. “What we need to be doing is trying to discover what triggers will convince the cells to do whatever it is that we want them to do.”
In the case of this experiment, the trigger is the combination of the BioDome and the drug cocktail. The BioDome is important because it provides a localized, controlled environment for the drugs to do their job. The drug cocktail, meanwhile, contained nothing leg-specific — which Levin said is of critical importance. Zero percent of the time did the experiment result in anything but leg creation.
“This technology is not about limbs per se; it is a way to look at and achieve regeneration of all kinds of body organs — anything that is missing, damaged, degenerating, cancerous, aging,” he said.
This strategy hinges on activating the information cells contain about creating body parts, as well as setting into motion natural regenerative abilities. After application of the BioDome containing the drug cocktail, the study team saw the activation of molecular pathways typically used when an embryo starts to take the shape of a body.
This activation is not necessarily because frogs are unique in their ability to regenerate limbs as juveniles, Levin said. Humans and many different animals have regenerative abilities. Children, for example, can regenerate lost fingertips, while adults experience constant regeneration on a smaller scale through the replacement of features such as skin, hair and intestinal lining.
The idea that mammals can regenerate through this intervention is being put to test by Levin and his colleagues in mice. He and co-author David Kaplan, a professor of biomedical engineering at Tufts University, have also co-founded a company, Morphoceuticals, with the aim to take these technologies toward clinical application. While people call asking to volunteer, the scientists are still far from human trials — Levin said it’s not possible to estimate when this will happen.
“I think it’s going to get there and I’m very optimistic about all of this,” he said. “But we’re not there yet. There’s some very important basic science that needs to happen before it’s ready to go on to a person.”
But in the far future, he can imagine the BioDome and its cocktail being applied to individuals in the hospital, and the concepts behind the work informing organ creation. If we know how to stimulate cells, then “we should be able to build whatever we want them to build,” Levin said.
The demand is already here. “People don’t realize the depth of biomedical suffering,” he said. “The need is profound.”