Scientists regenerate frog's lost leg.

The chance of restoring function through natural regeneration remains out of reach for millions of individuals who have lost limbs due to causes ranging from diabetes to trauma. Salamanders and superheroes are the only creatures who can regrow their legs and limbs.

Scientists from Tufts University and Harvard University's Wyss Institute released a study in the journal Science Advances that brings us one step closer to the aim of regenerative medicine.

The researchers used a five-drug cocktail administered in a silicone wearable bioreactor dome that seals in the elixir over the stump for just 24 hours to promote recovery of a severed leg in adult frogs who are naturally unable to regenerate limbs. After that quick therapy, an 18-month period of regrowth begins, restoring function to the limb.

Salamanders, starfish, crabs, and lizards are among the species that can fully regenerate at least some of their limbs. Flatworms may even be dismantled into individual pieces, with each piece reassembling an entire organism. Humans have the ability to close wounds with new tissue growth, and our livers have the amazing, almost flatworm-like ability to regenerate to full size after a 50% loss.

However, in humans and mammals, the loss of a large and structurally complicated limb, such as an arm or leg, cannot be recovered by any natural regeneration process. In fact, we often cover severe wounds with an amorphous mass of scar tissue to protect them from further blood loss and infection, as well as to prevent them from growing.

Getting the Regeneration Process 

Tufts researchers used a silicone cap called a BioDome to encase the wound in a silk protein gel infused with the five-drug cocktail, which activated the healing process in African clawed frogs.

Each medicine served a different objective, such as reducing inflammation, suppressing collagen formation that leads to scarring, and boosting new nerve fibre, blood vessel, and muscle growth. The bioreactor and the combination created a local environment and signals that tilted the scales away from the natural propensity to seal off the stump and toward the regenerative process.

Many of the treated frogs showed significant tissue growth, re-creating a nearly totally functional leg, according to the researchers. The new limbs contained bone structure that was comparable to that of a real limb, a larger complement of interior tissues (including neurons), and many "toes" that sprouted from the limb's end, albeit without the support of underlying bone.

The regrown limb moved and responded to stimuli such as a stiff fiber's touch, and the frogs were able to use it to swim through water and move like a normal frog.

"It's wonderful to see that the medications we chose were contributing to the creation of a nearly complete limb," said Nirosha Murugan, a research affiliate at Tufts' Allen Discovery Center and the paper's first author. "The fact that only a brief exposure to the medications was required to start a months-long regeneration process shows that frogs and possibly other animals have dormant regenerating capacities that can be put into action."

The researchers looked at the factors that could lead to long-term growth from a brief intervention. They observed the activation of known molecular pathways that are ordinarily used in a growing embryo to help the body take shape within the first few days following therapy.

Activation of these pathways could allow the limb to carry the weight of tissue growth and organisation, much like it does in an embryo, rather than requiring continual therapeutic intervention over the several months it takes to build the limb.

The BioDome in Action

The majority of animals that are capable of regeneration reside in an aquatic environment. The production of a blastema at the end of the stump, which is utilised to gradually restore the missing body part, is the initial step of growth after the loss of a limb. Within the first 24 hours following an injury, skin cells quickly cover the wound, safeguarding the reconstructing tissue beneath.

"Mammals and other regenerating animals will frequently have their injuries exposed to air or make touch with the ground, and they can take days to weeks to cover up with scar tissue," said David Kaplan, co-author of the study and Stern Family Professor of Engineering at Tufts. "Using the BioDome cap for the first 24 hours helps to simulate an amniotic-like environment, which, when combined with the correct medications, allows the rebuilding process to occur without hindrance from scar tissue."

Next Steps toward Frogs and  other Mammals regeneration

The Tufts researchers previously demonstrated that a single medication, progesterone, may cause considerable limb growth in the BioDome. The resulting limb, however, grew like a spike and was not the more typical shaped, functioning limb achieved in this work.

The five-drug cocktail is a major step forward in the restoration of fully functional frog limbs, and it suggests that further research into drug and growth factor combinations could lead to regrown limbs with even more functional features, such as normal digits, webbing, and more detailed skeletal and muscular features.

"Next, we'll see how this treatment works in mammals," said corresponding author Michael Levin, the Vannevar Bush Professor of Biology in the School of Arts & Sciences, director of the Allen Discovery Center at Tufts, and associate faculty member of the Wyss Institute.

"Covering the open wound with a liquid environment beneath the BioDome and using the proper pharmacological combination could deliver the crucial first signals to kickstart the regenerative process," he said. "It's an approach aimed at reactivating dormant, innate anatomical patterning programmes rather than micromanaging complex growth, because mature animals still retain the information they need to build their bodies."

Reference:

Nirosha J. Murugan, Hannah J. Vigran, Kelsie A. Miller, Annie Golding, Quang L. Pham, Megan M. Sperry, Cody Rasmussen-Ivey, Anna W. Kane, David L. Kaplan, Michael Levin. Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis. Science Advances, 2022; 8 (4) DOI: 10.1126/sciadv.abj2164