Bioreactor on the foot

Five signaling substances helped frogs grow severed paws

Polina Loseva, N+1

American scientists have learned how to start regeneration in adult frogs. To do this, they applied a hydrogel with signaling molecules to the place of the amputated paw. As a result, instead of a shapeless appendage, as usual, full—fledged paws grew in experimental animals - not fully formed externally, but absolutely functional. The work was published in the journal Science Advances (Murugan et al., Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis).

It is believed that once all four-legged vertebrates were able to regenerate well, but gradually most of them lost this ability. Now, only salamanders and tadpoles can easily grow new limbs for themselves. And in mammals, even cubs are almost unable to cope with this task — it is known that embryos in mice can restore a severed fingertip or a piece of heart tissue, but there is no question of whole body parts.

Therefore, scientists are looking for ways to force vertebrates to regain the lost abilities of their ancestors. A variety of methods are used: bioengineering frameworks, electrical stimulation, stem cell transplantation and processing with different signaling molecules. Michael Levin from Tufts University, together with colleagues, relied on the latter approach.

This group of researchers works with the spur frog Xenopus laevis — it is often used in regenerative studies, because this is one of those cases when a tadpole grows body parts quite well, and an adult loses this ability. 

In previous work, they found that the hormone progesterone stimulates regeneration quite well — however, it only helped to improve the growth of cartilage tissue itself, but not to restore the structure of the limb.

This is how a frog's paw grows without treatment (from above) and with progesterone treatment (from below). Herrera-Rincon et al. / Cell Reports, 2018.

Therefore, scientists decided to use several signaling molecules at once — to start the regeneration of all the tissues that make up the frog's paw. They assembled a cocktail of substances that were supposed not only to stimulate tissue growth, but also to restore the work of nerve cells and suppress inflammation. The result was a set of five substances: 1,4-DPCA, BDNF (brain neurotrophic factor), growth hormone, retinoic acid and resolvin D5. With solutions of these five substances, scientists impregnated a silk protein-based hydrogel, and a container with this hydrogel (BioDome) was sewn to the frogs to the wound surface. The signaling molecules worked for only 24 hours — after that, the container was removed. But this turned out to be enough for the researchers to notice the result, however, not immediately, but after a few months. Up to 6-8 months, limbs grew at a similar rate in those animals that received a signal cocktail, and in those who received a placebo container. But from the 9th month, the researchers recorded a difference, and the experimental group continued to overtake the placebo group up to 18 months of follow-up.

In the control group, which was not treated with anything, shapeless appendages formed in place of the limbs. In 20 percent of the frogs from the placebo group, scientists noticed something intermediate between shapeless tissue and a limb, but without signs of separation into areas characteristic of the hind leg. At the same time, 76 percent of the animals from the "cocktail group" have grown body parts resembling a paw: with a flattened end and webbing between the analogs of the fingers.

This is how the legs of frogs grew in the control group (upper row), with a placebo container (middle row) and when treated with signaling molecules (lower row). Murugan et al.

At the same time, apparently, the same thing happened in animals as in tadpoles. The latter, when they lose their limbs, first form a blastema — this is a tubercle of undifferentiated cells (which are obtained from differentiated ones). And then the blastema cells divide and re-specialize, turning into bone, muscle and skin cells. In frogs with amputated legs, under the influence of signaling substances, the wound closed more slowly (p<0.05) than in other groups in the experiment — which means that inflammation developed less in it, and the cells had more time to turn into a blastema. After the wound was closed, the tubercle still remained larger than in the other groups, and the cells in it expressed the main blastema marker SOX2 six times stronger than in the control group. After scanning the regenerated areas of the body, the scientists noticed a difference at the microscopic level. For example, the "cocktail group" turned out not only to have more bones by weight (p<0.05) — these bones were denser, and bumps and depressions appeared on the outside of them, which normally should serve to attach tendons and muscles. In addition, in this group, more vessels and nerve fibers were found in the regrown limbs. The sensitivity of these fibers, as the authors of the article found out, recovered to the level of a healthy limb. At least, most of the frogs from the "cocktail group" pulled back their regenerated paws at the slightest touch. The researchers also noticed that the animals used them to swim towards food or swim away from loud noises.

Reconstruction of bones in healthy (left) and regenerated (right, purple arrow) frog paws. On the left is an animal from the control group, in the center and on the right is an animal from the "cocktail group". Murugan et al.

The authors of the work urge to draw several important conclusions from their success. The first is that the success of regeneration is influenced by the microenvironment, and at the earliest stage. Even those animals that had a silicone placebo container sewn on grew parts of their paws more successfully than those who were left without a container. Perhaps the contact with the hydrogel itself slows down the closure of the wound on the amputated paw and triggers some important processes in the cells on the wound surface.

The second conclusion is that even in an animal that has lost the ability to regenerate, they can be restored or at least brought closer to the level at which they possess the early stages of its development. In this sense, the frog is, of course, a more convenient object than a mouse or a person — its larvae have these abilities well developed. Therefore, it may be more difficult to transfer these results directly to mammals. In addition, the researchers note, even a frog's paw grows for a long time — the experiment lasted a year and a half. It is not known how meaningful it is to try to grow limbs in humans at the same pace.

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