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Frogs Regrow Amputated Limbs After New Multidrug Treatment

regenerating frog limb photos
Limb regeneration in animals receiving no treatment (ND), BioDome only (BD), and BioDome with multidrug cocktail (MDT). | Murugan et al., Sci. Adv. 2022

A novel multidrug treatment delivered through a wearable device enabled African clawed frogs to successfully regrow their amputated hind legs, according to a new study in the January 28 issue of Science Advances. The 24-hour treatment resulted in functional limbs 18 months later that enabled the frogs to move around with natural ease.

The study demonstrates a technique to kickstart regeneration in an animal incapable of regrowing limbs on its own. By contrast, most previous limb regeneration studies have involved animals with natural regrowth capabilities. The findings could inform future studies to eventually explore regeneration in humans.

"No one can give a reliable estimate [of when this technique may be tested in humans] since we don't know how the science will go or what kind of funding will be available," said Michael Levin, a developmental and synthetic biologist at Tufts University and the corresponding author of the study. "But I think it has huge applications, because it's clearly possible to induce regeneration in a complex vertebrate which normally doesn't regenerate, without genome editing or gene therapy, and without heterologous stem cell implants."

"It's just a matter of time," he added. "We will crack this, for the benefit of many patients, and not just those with limb loss. This technique is general, for many organs."

Scientists have been eager to learn how to induce new patterned tissue to regenerate limbs for millions of patients, including diabetics and victims of trauma. But despite technological advances, doctors still lack tools to facilitate the recovery or reversal of tissue loss. Studies using model organisms have nonetheless provided insights, with many investigating the regenerative capabilities of the axolotl, (Ambystoma mexicanum), an ethereal salamander named for the Aztec god of fire and lightning that can regrow not just its limbs but also its heart, brain, and lungs.

However, Levin and colleagues have been inclined to study a model organism with more relatable limitations — the African clawed frog (Xenopus laevis), which can fully regenerate their hindlimbs as tadpoles but can only manage to rebuild featureless cartilaginous spikes by adulthood.

"These frogs are used for a lot of work in cancer, neuroscience, immunology, and birth defects," said Levin. "They have the same kind of limitations as humans with respect to regeneration as adults, so are a good model to try to turn it back on."

Guiding the body's 'build a leg here' decision

In November 2018, Levin and colleagues published a study using a bioreactor with progesterone to achieve some degree of limb regeneration in African clawed frogs, establishing a model for testing therapeutic cocktails to regenerate vertebrate hindlimbs. A few years later, in March 2021, Levin and colleagues published a study in Science Robotics on self-repairing synthetic living machines made from repurposed embryonic cells from the same frog species.

"Plus, we had done earlier work to trigger regeneration of tails and other organs in early tadpoles," said Levin. "So now we wanted to try it with adult animals, where no one had before shown the ability to regenerate."

To advance this research, Nirosha Murugan, a former postdoctoral researcher at Tufts University (currently an assistant professor at Algoma University) and the first author of the study, Levin, and colleagues amputated the hindlimbs of 115 adult female African clawed frogs and fitted them each with a "BioDome" device — a wearable bioreactor made from silk hydrogel. Devices in the test group were loaded with a novel five-drug cocktail designed to induce processes in the body such as modulation of inflammation, promotion of nerve regeneration, and tissue growth.

"[The device] does two things," said Levin. "It provides a controlled microenvironment in which the cells feel protected and safe to try to regenerate, and it allows us to use a silk gel to deliver a drug payload to the cells to push them towards the 'build a leg here' decision."

After 24 hours, the researchers removed the devices and returned the frogs to their tanks, where the amphibians swam around without further intervention aside from periodic checks on their progress over the next 18 months. Frogs that received the multidrug treatment showed a significant increase in soft tissue growth after only about two weeks. By nine months, the frogs showed increased tissue length relative to all other groups in the study — a leg up on the competition that they would sustain through the end of the trial. After 18 months, frogs that received the treatment also showed long-term soft tissue patterning and neuromuscular repair.

"On the one hand, we expected it to work, which is why we embarked on this long experimental program," said Levin. "On the other hand, seeing a year of regeneration after just 24 hours of treatment was incredible. It was profoundly moving to see it happening."

Together, the findings suggest that early, targeted interventions may overcome the need for ongoing therapies, such as the use of stem cells, that micromanage restorative growth and patterning. Instead, the study points to a more hands-off approach that kickstarts these processes in vertebrates.

"We are going to try this in mammals," said Levin. "We are already doing experiments in mice, using this and newer cocktails to try to understand how to kickstart organ regeneration."

[Credit for associated image: Celia Herrera-Rincon/Tufts University]

Author

Shannon Kelleher

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