Limb regeneration a reality: Scientists successfully regrow a frog’s lost leg

MEDFORD, Mass. — Scientists have successfully regrown a frog’s lost leg, bringing limb regeneration one step closer to reality.

Researchers from Tufts University say frogs – like humans – are naturally unable to regenerate limbs. However, scientists put a cocktail of drugs in a wearable dome, sealing the elixir over the missing limb for just 24 hours. This process set in motion an 18-month period of regrowth that restored the frog’s missing leg.

For millions of patients who have lost limbs for reasons ranging from diabetes to trauma, the possibility of natural regeneration remains out of reach. The groundbreaking therapy, described in the journal Science Advances, gives them fresh hope of a full recovery.

“It’s exciting to see that the drugs we selected were helping to create an almost complete limb,” says study first author Dr. Nirosha Murugan in a university release.

“The fact that it required only a brief exposure to the drugs to set in motion a months-long regeneration process suggests that frogs and perhaps other animals may have dormant regenerative capabilities that can be triggered into action.”

Mammals lack the ability to regenerate

Other creatures which have the ability to regenerate limbs include starfish, crabs, and some lizards. Flatworms can grow entirely new bodies even if something cuts them into several pieces. Unfortunately, humans are only capable of closing wounds with new tissue growth.

Our livers have a remarkable, almost flatworm-like capability of regenerating to full size after disease or injury destroys up to half of the organ. However, loss of an arm or leg is beyond any natural process of regeneration in humans or mammals.

Instead, scar tissue covers up major injuries, protecting the wound from further blood loss and infection — but preventing further growth. On the other hand, if a newt or salamander loses its leg, it will grow another. The wound will close and, over time, it will create new bones, muscles, nerves, and skin.

In this study, researchers triggered the regenerative process in African clawed frogs by enclosing the wound in a silicone cap – called a BioDome. It contained a silk protein gel loaded with five drugs, each fulfilling a different purpose. Scientists dampened inflammation and inhibited production of collagen which causes scarring. They also encouraged new nerve fibers, blood vessels, and muscle to form.

The bioreactor provided a local environment and signals that tipped the scales away from the natural tendency to close off the stump and, instead, move towards the regenerative process. Researchers observed dramatic growth of tissue in many of the treated frogs — re-creating an almost fully functional leg.

New leg just like the old leg

The new limbs had bone structure extended with features similar to their natural leg. They also had a richer complement of internal tissues, including neurons. Several “toes” grew from the end of the limb, although without the support of underlying bones. The regrown limb moved and responded to stimuli such as a touch from a stiff fiber.

The frogs were also able to make use of their new leg for swimming through water, moving much like the one on the opposite side.

Study authors explored the mechanisms by which the brief intervention could lead to long-term growth. Within the first few days after treatment, they detected the activation of known molecular pathways that are normally used in a developing embryo to help the body take shape. Activation of these pathways could allow the burden of growth and organization of tissue to be handled by the limb itself.

This is similar to how it occurs in an embryo, rather than requiring ongoing therapeutic intervention over the many months it takes to grow the limb. Animals naturally capable of regeneration live mostly in an aquatic environment.

Stem cells play a key role in regeneration

The first stage of growth after loss of a limb is the formation of a mass of stem cells at the end of the stump, called a blastema. It gradually reconstructs the lost body part. The wound is rapidly covered by skin cells within the first 24 hours after the injury, protecting the reconstructing tissue underneath.

Mammals and other regenerating animals will usually have their injuries exposed to air or making contact with the ground, and they can take days to weeks to close up with scar tissue,” says co-author Professor David Kaplan.

“Using the BioDome cap in the first 24 hours helps mimic an amniotic-like environment which, along with the right drugs, allows the rebuilding process to proceed without the interference of scar tissue.”

Moving from one drug to a regeneration cocktail

Previous work by the same group showed a significant degree of limb growth thanks to a single drug, progesterone, with the BioDome. However, the resulting limb grew as a spike and was far from the more normally shaped, functional limb achieved in the current study.

The five-drug cocktail represents a significant milestone toward the restoration of fully functional frog limbs. It suggests further exploration of drug and growth factor combinations could lead to limbs that are even more functionally complete. They would have normal digits, webbing, and more detailed skeletal and muscular features.

“We’ll be testing how this treatment could apply to mammals next,” adds corresponding author Prof. Michael Levin, director of the Allen Discovery Center at Tufts.

“Covering the open wound with a liquid environment under the BioDome, with the right drug cocktail, could provide the necessary first signals to set the regenerative process in motion,” Levin concludes. “It’s a strategy focused on triggering dormant, inherent anatomical patterning programs, not micromanaging complex growth, since adult animals still have the information needed to make their body structures.”

South West News Service writer Mark Waghorn contributed to this report.

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