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.

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).

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.

For more information:

• “With some help from salamanders, will humans one day be able to regrow limbs?” – STAT
• “If other animals can regenerate their limbs, why can’t humans?” – Public Radio International