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

  1. Looking to the future:
    What we are is a long way from ethical code of conduct in the scientific animal studies. (Repeat amputation?) I understand how this particular study has benefited a potential to future human medical procedures, but have we absolutely no conscience at all? What kind of a person could design a study that inflicts repeated harm on an animal? There must be a smarter way. I would never be so naive to think that we know everything about how animals experience pain.
    Forgive me. For as interesting the discovery, and well-written the article, I found myself reacting pitifully to the barbaric treatment of a living creature that I know is widespread in the scientific world. May we open & evolve to have the wisdom to contain our urges; seeking to know everything at the cost of the innocent reveals just how irresponsible and foolish we really are.

    1. You rightly point out that we probably know very little about how animals experience pain, and that repeated amputation must be a horrible experience to be subjected to (the word “torture” comes to my mind, even though the scientists does not do it for fun). You also say that “there must be a smarter way”, but here I have to disagree with you, currently there is no smarter way (as far as I am aware). There are many people that are critical about the use of animals in experiments, but at the same time they enjoy the advantages that came from such experiments. What is more, aside from voicing their oppinins about the use of animals in experiments, they do not come up with alternatives. Indeed we must “evolve” , and perhaps if you can come up with an alternative to animal models, we can proceed on that trajectory.

      1. Very sensible and well-put comment; I totally agree! Knowledge and discovery come at a price; and new discoveries deserve proper respect and responsible use.

    2. You can easily monitor it among a population of them or from breeders. The shop I purchased mine from has tons of babies and since they do not have their sense of smell yet many of the babies do not have arms, legs or tails because the alpha babies will eat the the smaller ones limbs if it is in front of their mouths . There will be one or two that start to grow a lot faster than the others because they eat the food and the smaller axolotl’s limbs over and over again until you separate them but then one or two will take their place and grow larger and so on and so on.

    3. You are insane to say the least..
      Research like this helps the human race to have a better future.
      May if you lose a limb
      you will realise how much this ” barbaric act ” will help people in future
      As for alternative way , doing research is extremely difficult and pretty often there is dead end to any procedure.
      So whatever options we have we should cling onto it..

  2. Crippled children before these little critters till we learn or relearn how to do it on our own. Let the children thrive

    1. You can buy them online but you pay for express shipping. Many local pet store can get their hands on them they are a handful but easy to take care for if you just make sure your axolotl is in brackish water. Also make sure its an axolotl and not a mud puppy they look exactly the same but axolotls never mature and stay in a juvenile state while a mud puppy will mature into a salamander.

      1. Do NOT put the axolotl in brackish water. It comes from a freshwater lake.

        They require cold water. Around 65 degrees. Thats about it. Good filtration and a cycled aquarium.

  3. As an owner of an Axolotl the regeneration ability of them are amazing. My axolotl lost five of his six gills due to a fungi infection and they have fully regrown in 5 days. They will also eat their own arms, legs and tails to keep themselves fed with their limbs growing back quickly. When my axolotl was growing he ate his tail and it regrew in a week once I found the amount of food he required. Chimera axolotls are amazing because they will be split right down the middle two different colors and can be both male and female depending on which embryos merge.

    1. I have a few and would like to try and breed them. I was told that I need to gradually drop the temperature and eventually raise the temperature back to 18, thereby mimicking what happens in nature. this prompts the axolotls to reproduce. How can this be achieved practically? Thanks

      1. I was under the impression that once they reached maturity they did it naturally and you didn’t have to do anything? How old are your axolotls? They usually reach sexual maturity at around 12-14 months

    2. I completely agree! I would love a Chimera axolotl, especially if it was partially leucistic, partially golden albino.

  4. For as interesting the discovery, and well-written the article, I found myself reacting pitifully to the barbaric treatment of a living creature that I know is widespread in the scientific world. May we open & evolve to have the wisdom to contain our urges; seeking to know everything at the cost of the innocent reveals just how irresponsible and foolish we really are.

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