by Garrett Dunlap
figures by Rebecca Senft

Limb loss affects nearly 2 million people in the United States alone. While many instances are related to traumatic events like car accidents, the majority of limb loss cases are caused by diseases that affect the body’s blood vessels. One such disease is diabetes, in which gradual declines in blood flow to a patient’s lower extremities can eventually lead to loss of the entire limb. If the incidence of diabetes continues to rise, there will likely be a corresponding increase in the number of people who must confront limb amputation. Unfortunately, the current therapeutic options following amputation are not much changed from centuries ago, with prosthetic limbs remaining the only option for replacement. But while replacement artificial limbs have been able to replace the form of the lost limb, their function remains severely lacking, especially when the lost appendage is an entire arm or leg. So what if instead of relying upon a wooden or metallic impostor, we might one day just regrow a lost limb?

Many animals have the power of regeneration

To begin thinking about how to accomplish human limb regeneration, scientists have taken note of animals that already show this ability. A prime example is the axolotl (Ambystoma mexicanum), a species of aquatic salamander. Unlike humans, it has the “superpower” of regenerating its limbs, spinal cord, heart, and other organs. But the axolotl is not the only member of the animal kingdom that can do this (Figure 1), as many invertebrates (animals without a spine) are masters of regeneration. Flatworms and hydra, for instance, can regrow their entire bodies from only a tiny piece of their original selves. Even among vertebrates (animals that do have spines), the axolotl isn’t the only animal capable of regeneration. Young frogs are known to regrow limbs, though they lose this ability when they change from tadpoles to adult frogs. On the other hand, the axolotl retains it throughout its entire life, making it unique among vertebrates and a great model to study in regeneration research.

Figure 1: Many animals undergo regeneration (at least to some degree). While the axolotl is not the sole master of regeneration in the animal kingdom, it is the only vertebrate that can regenerate many body parts throughout its entire life.

While there are no known mammals that can fully regenerate missing appendages, many harbor hints of regenerative potential—humans included. It has been observed that mice can regenerate the tips of their toes, though loss further up the foot results in the same scarring that humans see after amputation. Humans have also been known to regenerate the tips of the fingers, including the bone and skin. Multiple clinical reports in the past decades have documented such instances following traumatic injury. Unfortunately, this response gets weaker as the site of loss occurs closer to the palm. While this ability has undoubtedly helped some people in the event a traumatic injury, it is a far cry from the axolotl’s ability to regenerate a fully-formed limb with all of its normal muscles, cartilage, and other tissues.

How does regeneration work?

In axolotls, the process that results in regeneration of an entire limb (Figure 2) involves a complex orchestration of the limb’s surviving cells. Following limb loss (B), a clot of blood cells rapidly stops bleeding at the cut site. After this, a layer of cells works to quickly cover the plane of amputation, forming a structure called a wound epidermis (C). During the next few days, the cells of the wound epidermis grow and divide rapidly. Shortly thereafter, the cells underneath the epidermis also begin to rapidly divide, forming a cone-shaped structure known as a blastema (D). The cells that make up the blastema are thought to be bone, cartilage, muscle, or other cells that de-differentiate (lose their identity) to become similar to stem cells, which are cells that can become one of many different kinds of cells. Blastema cells, however, have restrictions on the types of cells that they can become: for instance, a blastema cell that used to be a muscle cell can only re-form different types of muscle cells, not skin or cartilage cells. These de-differentiated cells in the blastema then grow and multiply, eventually regaining their identity as fully-developed bone or skin cells (E). As the blastema and its cells continue to divide, the growing structure flattens and eventually resembles a perfect copy of the lost limb, including nerves and blood vessels that are connected to the rest of the body (F).

Figure 2: Axolotl limbs go through a multi-stage process following injury to regenerate the lost appendageSkin, bone, cartilage, and muscles can be regrown many times with no sign of trauma.

Learning from the axolotl

To even begin to think about how we can one day be able to regrow lost human limbs, scientists must become intimately familiar with the changes that axolotl cells undergo during regeneration. One approach that has been successful thus far is discovering molecular tweaks that cause an axolotl to lose its regenerative ability, which can reveal regeneration’s most important components and contributors. For instance, the immune system was found to be an important player the limb regeneration process. Macrophages, which are cells that serve a critical role in the inflammation response after injury, were previously connected to regeneration. In fact, injecting a drug to get rid of macrophages in an axolotl’s limb before amputation leads to the accumulation of scar tissue instead of regrowth. This scarring, which happens when a protein called collagen becomes disordered, is a normal part of wound healing in humans, but it is unusual in axolotls. This result suggests that macrophages may be essential for regeneration. Tweaking the nervous system has also been shown to interfere with regeneration. Scientists have observed that surgically removing a limb’s nerves prior to amputation can hinder regeneration, though work is still being completed to better understand why this happens.

All of these previous methods, though, rely on needing to remove an otherwise crucial part of a healthy body (e.g. immune cells and parts of the nervous system). But scientists are now diving down to the level of genes to search for new insights. To accomplish this, researchers first attempted to answer the question of how many times an axolotl limb can successfully regenerate. By repeatedly amputating limbs, it was seen that by the fifth time, few limbs could regrow to their previous potential. Further, when the limbs that could not regenerate were studied further, researchers again found extensive scar tissue build-up, paralleling what is often seen in human injuries. By comparing the genes that were turned on or off when the axolotl’s limb wasn’t able to regrow, scientists have found more molecules and processes to study that hold promise for kick-starting regeneration in humans. Perhaps one day, drugs can be made to modulate these genes, causing them to turn on and help a human limb to regrow after amputation.

Looking to the future

While we are still a long way from regrowing a human limb, we place ourselves at a disadvantage if we lack an understanding of how regeneration occurs in the lucky animals that already hold this “superpower.” Aided by tools that allow scientists to see the fine genetic details of the regeneration process, we are slowly inching closer to understanding what makes regeneration tick. To test this, scientists are working diligently to develop new tools that will allow them to identify other targets and begin transferring these insights to mammals like mice, meaning that perhaps one day, the millions living with lost limbs will have a new avenue for treatment: regeneration.

Garrett Dunlap is a student in the Biological and Biomedical Sciences Ph.D. program at Harvard University.

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83 thoughts on “Regeneration: What the axolotl can teach us about regrowing human limbs

  1. When I was young and working in our garden by our pond I happened to see a monster green bull frog along the bank. Thinking it would be good in our frying pan I snuck up on it and hit it with a garden hoe nearly severing one of it’s back legs above the knee joint. It somehow managed to get back in the water and get away. About a month later I saw this same frog along the bank of the pond with the same damaged leg that was rotted and just hanging on by some skin with a new shorter leg growing where it should be.

  2. I came here to learn about Axolotls as pets, but this was far more interesting than I expected!

  3. I hate to sound like I know it all ..but I actually do know why humans can can’t grow limbs back…. It’s because none of us humans believe that we can… Because we have the ability and the freedom to think positively or negatively… On this subject we’ve been taught through generations that we cannot grow limbs… It’s in our DNA and will be overcome in future generations… Our DNA contains the sum total of thoughts wisdom or evolution however you would like to put it. … , We humans can Manifest anything that we believe we can… Including limbs of course… Sadly, most scientists don’t understand that a belief is only a thought we have over and over again. And that we can change our beliefs eventually so that we can manifest anything….

    1. Please do a LITTLE research on mRNA and tell me you still believe it’s possible to just: ‘think hard enough and regenerate limbs’ …Correct me if I misunderstood but I’m pretty sure that’s what you are saying.

  4. After reading several of these articles. I’m not surprised that people react as they do.
    Whoever said that life was fair, just, and equally divided or dispersed among everyone through life or anyone’s life. Should have learned the answer to that as far back as 2008 in this country. That’s another topic but not for now.

    Without such science where would be today? I would think many years in the past still today wondering, what do we do now?
    Maybe if you were an amputee as I am. Maybe the use of science would be a little more understanding.
    How does one put such empathy on an animal and still drive a vehicle? How many people have been cripples or killed by an automobile each day? Yet we drive them anyway. No one stands on a street corner with a sign saying, vehicles are bad for you!

    Don’t think I agree with cruelty at all. There is a big difference between that and science.
    We just have to say to ourselves that is another necessity in life and just say thank you to those that do the world a service.
    I would hope to think they don’t go home and feel happy about an animal losing its life for no reason. Now think about war. Put your empathy out there for those special people that gave all for you so you could feel sorry for an animal.
    They would love to be today what they were before they left to war for you. Hence, I hope science develops a way to grow my limb back. I could shake your hand again.

    1. Amen, so glad someone sees the practical side of the article and isn’t just trying to turn all of us down a guilt trip path…of course, that’s how a lot of people argue these days, instead of reasoning rationally and considering the feelings of those who are actually amputees, they just talk about (correct me if I misunderstood) going vegetarian and avoiding large sources of protein to cure basically anything.(Ahem, Ariana Boes) Huh, right. let me know when you do a little actual research on diseases like diabetes and also when you lose an arm like Tom C over here…you might feel different then.

    2. A veteran, Tom C, correct? if so, thank you for your service and the same to all the heroes who do the same so we can ‘feel sorry for an animal’. Very well put. I hope you do get your limb back, because I would like to shake your hand.

    3. I understand your pain, but if we destroy the ecosystem, we will all be equally extinct. You are crippled, but also lucky. Don’t throw away your chance, next time you won’t live.

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