Scientists regrow a missing frog leg with a cocktail of five drugs

Frogs treated briefly with a five-drug cocktail administered by a wearable bioreactor on the trunk were able to regrow a nearly fully functional limb.

For the millions of patients who have lost limbs from causes ranging from diabetes to trauma, the possibility of restoring function through natural regeneration remains elusive. Regrowing legs and arms remains the prerogative of salamanders and superheroes.

But in a study published in the journal science advances, Scientists at Tufts University and the Wyss Institute at Harvard University are one step closer to achieving the goal of regenerative medicine.

For adult frogs, which naturally cannot regenerate limbs, the researchers were able to regrow the lost leg using a mixture of five drugs placed in a wearable silicone bioreactor dome that seals the elixir over the stem for just 24 hours. This short treatment begins an 18-month period of regrowth that restores a functional leg.

Many organisms have the ability to fully regenerate at least some limbs, including salamanders, starfish, crabs, and lizards. Flatworms can be cut into pieces, each piece rebuilding an entire organism. Humans are able to close wounds with the growth of new tissue, and our livers have a remarkable, nearly flatworm-like ability to regenerate to full size after a 50% loss.

But the loss of a large and structurally complex limb – an arm or a leg – cannot be compensated for by any natural process of reproduction in humans or mammals. In fact, we tend to cover large injuries with an amorphous mass of scar tissue, which protects them from further blood loss and infection and prevents further growth.

African clawed frog

Ordinary African clawed frog. Credit: Busen Oliver

Renewal

Tufts University researchers triggered the regeneration process in African clawed frogs by coating the wound with a silicone sleeve, which they call the BioDome, which contains a silk protein gel loaded with a five-drug cocktail.

Each drug served a different purpose, including reducing inflammation, inhibiting collagen production that may lead to scarring, and encouraging new growth of nerve fibers, blood vessels, and muscle. The collection and bioreactor provided a local environment and signals that shifted the scales away from the natural tendency to close the stem, and toward the regeneration process.

The researchers observed significant tissue growth in several of the treated frogs, which reconstituted a nearly fully functional leg. The new limbs had an extended bone structure with features similar to that of normal limb bone structures, a richer complement of internal tissues (including neurons), and several ‘fingers’ grew from the end of the limb, despite the lack of support from the underlying bone.

The regrown limb moved and responded to stimuli like a touch of a hard fiber, and the frogs were able to make use of it to swim in the water, moving like an ordinary frog.

frog limb regeneration

The soft tissues of MDT animals were consistently longer than that of BD or ND of 8 MPa [F(2,19) = 61.9, P < 0.05]. Credit: Murugan W. the. , Science Advance 2022, DOI: 10.1126 / sciadv.abj2164

“It is exciting to see that the drugs we chose help form a near-complete limb,” said Nerusha Murugan, research firm at the Allen Discovery Center in Tufts and first author of the research paper. “The fact that it required only a short exposure to the drugs to initiate the regeneration process for months suggests that frogs and possibly other animals may have dormant regeneration capabilities that can be activated.”

The researchers explored the mechanisms by which a short intervention could lead to long-term growth. Within the first few days after treatment, they discovered activation of known molecular pathways that are normally used in a developing fetus to help the body form.

Activation of these pathways could allow the burden of growth and tissue regulation to be handled by the limb itself, similar to the way it occurs in a fetus, rather than requiring continuous therapeutic intervention over the many months it takes for a limb to grow.

How does BioDome work

Animals that are naturally able to regenerate mostly live in an aquatic environment. The first stage of growth after the loss of a limb is the formation of a mass of stem cells at the end of the stem called the blastema, which is used to gradually rebuild the lost part. The wound is quickly covered by skin cells within the first 24 hours after injury, protecting the reconstructed tissue underneath.

“Typically, mammals and other animals that regenerate are exposed to air or contact with the ground, and can take days to weeks to close with scar tissue,” said David Kaplan, professor of engineering in the Stern family at Tufts University and colleagues. Study author. “Using the BioDome cap in the first 24 hours helps simulate a fetal-like environment which, combined with appropriate medications, allows the reconstruction process to proceed without the involvement of scar tissue.”

Next steps in frogs and mammals

Previous work by the Tufts team demonstrated a significant degree of limb growth induced by a single drug, progesterone, with the BioDome. However, the resulting tip grew in the shape of a nail and was far from the functional, normal-shaped tip achieved in the current study.

The five-drug cocktail represents an important milestone toward restoring fully functional frog limbs and suggests further exploration of drug groups and growth factors that could lead to regrowth of limbs that are more fully functional, with normal toes, girdle, and more detailed skeletal muscle features.

Corresponding author Michael Levine, professor of biology at Vannevar Busch School of Arts and Sciences, director of the Allen Discovery Center at Tufts, and associate faculty member at the Wyss Institute said.

“Covering the open wound with a liquid environment under the BioDome, with the appropriate drug cocktail, can provide the initial signals needed to start the regeneration process,” he said. “It’s a strategy that focuses on unlocking the underlying and inherent anatomical modeling software, not the detailed management of complex growth, because adult animals still have the information needed to make up their body structures.”

Reference: “Acute Multidrug Delivery via a Wearable Bioreactor Facilitates Long-Term Limb Regeneration and Functional Recovery in Adult Xenopus laevis” By Nerusa J. Morgan, Hannah J. Vigran, Kelsey A. Miller, Annie Golding, Quang L Pham, Megan M. Sperry , Cody Rasmussen Ivy, Anna W. Kane, David L. Kaplan and Michael Levine, January 26, 2022, science progress.
DOI: 10.1126 / sciadv.abj2164

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