Drug Cocktail Enables Frogs To Regenerate Amputated Limbs

Researchers have developed a multidrug-loaded wearable bioreactor that can trigger the restoration of amputated hindlimbs in adult African clawed frogs (Xenopus laevis). The animals were able to regrow and remodel a missing limb and sensorimotor function was restored.

Until now, most work focused on limb regeneration has been performed using model organisms with natural regrowth capabilities and there has been limited success regrowing new functional limbs in nonregenerative adult models. The findings of this new study, published in Science Advances, could be a “leap” towards developing limb regeneration approaches in humans.

Despite technological advances, the underlying mechanisms driving limb regrowth remain unclear. The study’s corresponding author Prof. Michael Levin, principal investigator of the Levin Lab at Tufts University, elaborates in an interview with Technology Networks, “There are many molecular players (e.g., specific signaling factors and genes) known to be involved in (and necessary for) limb growth and regrowth (in animals that regrow them, such as salamanders). It is not really known what ‘drives’ limb regrowth.”

Various techniques have been explored in efforts to induce limb regrowth in animals, including electrical stimulation, tissue-guiding biomaterials, progenitor cell transplantation and up-regulation of specific molecular pathways. However, according to Levin these approaches almost always use small froglets ‒ very young/immature animals that already have a degree of regenerative capacity.

“We’ve used very large, adult animals which is unique ‒ they regenerate very poorly, which makes it more challenging to do and more impactful if it works,” says Levin. “Our strategy uses a new cocktail of drugs that has never been used before, and also delivers it via a wearable bioreactor. There are no stem cell implants or foreign genes introduced (as in some other approaches).”

Building, installing and removing the bioreactor

The first step for Levin and his team was to develop a wearable bioreactor device capable of controlling the local microenvironment of the amputation site. The device was composed of an insert containing a silk hydrogel designed to carry substrates and release them in a controlled manner.

The plan was to then “load” the bioreactor (named BioDome) with small-molecule compounds (the substrate) known to encourage cells toward the program of making a limb, as opposed to micromanaging the process using stem cell 3D printing approaches. “The idea is to find a trigger, not to implement all the details of growing a limb. This is why our approach uniquely involved 24 hours of treatment followed by well over a year of growth ‒ the goal is to capitalize on the ability of the cells themselves to build complex structures,” notes Levin.

The adult female African clawed frogs were randomly allocated to one of three treatment groups: No device (control), BioDome only or BioDome plus drug cocktail. The authors noted that “the control animals were handled similarly to those that received devices, but no devices were attached to the wound stump.”

For the groups fitted with the device, exposure was limited to a 24-hour period post-amputation. Levin explains that the exposure was limited to 24 hours because the point of the study was to trigger regrowth at the site, rather than provide the animals a long-term micromanagement strategy. “We don't know if the 24-hour window can be shortened ‒ perhaps it can be even shorter. In past work, we showed that tail regeneration can be turned on by just 1-hour exposure to a bioelectric drug,” notes Levin.

Upon removal of the device, the animals were maintained for 18 months, and limb regrowth was followed during this period.

Choosing the “right” drug cocktail

The drugs were selected for specific effects that the team thought would trigger pro-regenerative responses very early on, when the frog’s cells at the site of amputation decide whether to embark on limb regrowth or not. “The intervention contained 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid, brain-derived neurotrophic factor (BDNF), growth hormone (GH), resolvin D5 and retinoic acid,” says Levin. The authors outline the reasons for choosing each of the compounds in the paper’s discussion section:

• BDNF delivery to nonregenerative rodents has been shown to increase the number of dorsal root ganglion neurons and peripheral axon and also encourages recovery of motor functions
• GH supplementation has been shown to restore animals’ ability to regenerate limbs in those without a pituitary
• 1,4-DPCA inhibits the excessive deposition of collagen and is an inducer of processes known to encourage the formation of new blood vessels
• Resolvin D5 promotes anti-inflammatory responses and also reverts inflammatory responses back to a noninflamed state.
• Retinoic acid is a morphogen (a signaling molecule know to act over long distances) and has been established as a key player in vertebrate limb development.

Why are African clawed frogs an ideal model system?

According to Levin, these animals are ideal because, like humans, and unlike other popular model organisms such as the salamander or flatworm, they have limited regeneration capacity. Levin elaborates on their suitability: “They are complex vertebrates, with large limbs onto which bioreactors can be attached and have functional endpoints (sensing and moving the limb) that can be used to ascertain the quality of the regenerate.”

However, he also notes one key limitation – time. “The regeneration process takes many months, therefore it’s not a rapid system for approaches such as drug screening.”

Levin concludes by touching on future research plans: “We are going to try this strategy (and our other strategies) in mice to better understand how rapid signals can trigger organ-building subroutines.”

Reference: Murugan NJ, Vigran HJ, Miller KA, et al. Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis. Sci. Adv. 2022. doi: 10.1126/sciadv.abj2164

Michael Levin was speaking to Laura Elizabeth Lansdowne, Managing Editor for Technology Networks.