Tag Archives: limb

Researchers successfully regrow limbs on frogs. They want to do the same thing with humans

Most animals have pretty good injury repair capabilities, but when it comes to lost limbs, only a select few can regrow them. The rest, including humans, have little they can do to repair such injuries. But as a new study shows, with the right treatments, our bodies may be hacked and “convinced” to regrow lost limbs. Although the study focused on frogs, which are obviously very different from humans, the proof-of-concept study suggests that this approach could work on many animals, including humans.

The African clawed frog (Xenopus laevis). Image via Wiki Commons.

Limb regeneration is a new frontier in biomedical science. It’s something we’ve long considered outside the realm of possibility, restricted only to superheroes and myth, but research is bringing it closer and closer to reality.

While many things differentiate humans from frogs, neither we nor they are able to regenerate limbs. So researchers at Tufts University and Harvard University’s Wyss Institute used frogs (specifically, the African clawed frog or Xenopus laevis) as a proof of concept. X. laevis is often used in research as it is easy to handle, lays eggs throughout the year, and for a model organism, shares a close evolutionary relationship with humans.

The researchers triggered the regrowth of a lost leg using a five-drug cocktail that they applied in a wearable silicone bioreactor dome that sealed the drugs over the stump for just 24 hours. After the treatment was administered, the regenerative process was kickstarted, and over the course of an 18-month period, the frogs regrew an almost fully functional leg.

“It’s exciting to see that the drugs we selected were helping to create an almost complete limb,” said Nirosha Murugan, research affiliate at the Allen Discovery Center at Tufts and first author of the paper. “The fact that it required only a brief exposure to the drugs to set in motion a months-long regeneration process suggests that frogs and perhaps other animals may have dormant regenerative capabilities that can be triggered into action.”

The experiment was repeated on dozens of frogs, and while not all of them regrew limbs, most did — including bone tissue and even toe-like structures at the end of the limb (though these weren’t supported by bone). It’s not a magic elixir, and the treatment is not perfect, but the drug cocktail delivered through the wearable bioreactor really does seem capable of regrowing limbs.

Regrowth of soft tissue. The MDT group (bottom) represents the five-drug cocktail treatment. Image credits: Murugan et al (2022).

The researchers essentially hacked the biological pathways that enable the growth and organization of tissue — much like in an embryo. This is why the treatment was only applied once, over the course of a day; meanwhile, other approaches involve numerous interventions over the course of the process.

“The remarkable complexity of functional limbs suggests that the fastest path toward this goal may lie in triggering native, self-limiting modules of organogenesis, not continuous micromanagement of the lengthy process at the cell and molecular levels,” the researchers write in the study. “We implemented this via a short exposure of limb amputation wounds to a wearable bioreactor containing a payload of five select biochemical factors.”

The first stage is the formation of a mass of stem cells at the end of the stump, which was then used to gradually reconstruct the limb. It’s essential that this structure is covered with the dome as quickly as possible after amputation to ensure its protection and activation. This treatment would be ideally applied right after amputation.

“Mammals and other regenerating animals will usually have their injuries exposed to air or making contact with the ground, and they can take days to weeks to close up with scar tissue,” said David Kaplan, Stern Family Professor of Engineering at Tufts and co-author of the study. “Using the BioDome cap in the first 24 hours helps mimic an amniotic-like environment which, along with the right drugs, allows the rebuilding process to proceed without the interference of scar tissue.”

At first, researchers tried using the protective dome with a single drug, progesterone. Progesterone is a steroid hormone involved in the menstrual cycle, pregnancy, and embryogenesis of humans and other species. This alone triggered some limb growth, but the resulting limb was essentially a non-functional spike. Each of the other four drugs fills a different role, ranging from reducing inflammation and the stopping of scar tissue formation to the promotion of growth of new nerves, blood vessels, and muscles. It’s the combination of all these together that leads to a nigh-functional limb.

Researchers note that while the limbs weren’t 100% identical to “normal” limbs, they featured digits, webbing, and detailed skeletal and muscular features. Overall, the results show the successful “kickstarting” of regenerative pathways

The plan now is to move on to mammal research. Despite the differences between frogs and mammals, researchers say that the biggest difference lies in the “early events of wound healing” — if these early processes can be understood and replicated, then there’s no apparent reason why this couldn’t be applied to mammals, and ultimately humans as well.

“The goal of triggering latent tissue-building routines to regrow limbs in humans may be achieved by identifying and exploiting principles observed in highly regenerative organisms,” the researchers conclude.

The study was published in the journal Science Advances.

Frog pre-amputation.

Experimental bioreactor helps frogs regenerate lost limbs

We’re one leg closer to developing functional limb regeneration.

Frog pre-amputation.

Xenopus laevis pre-amputation
Image credits Celia Herrera-Rincon / Tufts University.

A team of researchers from the Tufts University wishes that everyone could lose a foot and have it, too. The group successfully “kick-started” partial tissue regrowth in adult African clawed frogs (Xenopus laevis) through the use of a bioreactor and electroceutical (electrical cell-stimulating) techniques.

The cradle of life

“At best, adult frogs normally grow back only a featureless, thin, cartilaginous spike,” says senior author Michael Levin, developmental biologist at the Tufts University’s Allen Discovery Center.

“Our procedure induced a regenerative response they normally never have, which resulted in bigger, more structured appendages. The bioreactor device triggered very complex downstream outcomes that bioengineers cannot yet micromanage directly.”

The scientists split up the frog models into three groups — one experimental, one control, and one ‘sham’ group. Each animal had one of its hindlimbs amputated for the trial. Next, they 3D printed a “wearable bioreactor” out of silicon and filled it with hydrogel (a tissue-like mix of water and polymers). This hydrogel was mixed with certain silk proteins that provided a “pro-regenerative environment” and “enhanc[ed] bone remodeling”, according to the authors.

Next came the trial proper: frogs in the experimental and sham groups received the bioreactor (which was sutured on) immediately after the amputation procedure. The difference between the two is that the hydrogel for the experimental group was further laced with progesterone. Progesterone is a hormone that works to prepare the body for pregnancy but has also been shown to promote tissue repair, from nerves to bone. The control group received no treatment. Twenty-four hours later, the devices were removed.

Observations carried out at various times over the following nine-and-a-half months show that the bioreactor induced a degree of regeneration in the experimental group that had no counterpart in the other two groups. Instead of the typical spike-like structure, frogs treated with the bioreactor-progesterone combo re-grew a paddle-like structure — closer to a fully formed limb than what unaided regeneration processes created.

Results comparison.

Image credits Celia Herrera-Rincon et al., 2018, Cell Reports.

“The bioreactor device created a supportive environment for the wound where the tissue could grow as it did during embryogenesis,” says Levin. “A very brief application of bioreactor and its payload triggered months of tissue growth and patterning.”

The regenerated structures of the experimental groups were thicker, had better-developed bones, nerve bundles, and blood vessels. Video footage of the frogs in their tanks also showed that these frogs could swim more like un-amputated ones, the team adds. Scarring and immune responses were also dampened in the bioreactor-treated frogs, suggesting that the progesterone limited the body’s natural reaction to injury in a way that benefited the regeneration process.

So, why exactly did the device work? Genetic tests performed by the team showed that the bioreactor-progesterone combo altered gene expression in cells at the amputation site. Genes involved in oxidative stress, serotonergic signaling, and white blood cell activity were upregulated, while some other signaling-related genes were downregulated.

Regeneration results 2.

Anatomical outcome (bottom) and X-ray images (top) of regenerates formed in adult Xenopus hindlimb amputation after no treatment (Ctrl, A) and after 24-hr combined treatment of drug-loaded device (Prog-device, B–D).
Image credits Celia Herrera-Rincon et al., 2018, Cell Reports.

“In both reproduction and its newly discovered role in brain functioning, progesterone’s actions are local or tissue-specific,” says first author Celia Herrera-Rincon, neuroscientist in Levin’s lab at Tufts University.

“What we are demonstrating with this approach is that maybe reproduction, brain processing, and regeneration are closer than we think. Maybe they share pathways and elements of a common — and so far, not completely understood — bioelectrical code.”

The team plans to expand their research in mammal subjects. Previous research hinted that mice can partially regenerate tissue (such as amputated fingertips) under the right conditions. Life on land, however, hinders this process. “Almost all good regenerators are aquatic,” Levin explains, adding that “a mouse that loses a finger or hand, and then grinds the delicate regenerative cells into the flooring material as it walks around, is unlikely to experience significant limb regeneration.” Still, let’s keep our fingers crossed that the team finds an elegant and efficient solution to this problem — it may, after all, be our limbs that we regrow one day!

But there’s much work to be done until then. Levin says the next step is to add sensors to the device for remote monitoring and optogenetic stimulation, which should give the team a degree of control over how tissues regenerate in the bioreactor. They also plan to expand on their work with bioelectric processes in the hopes of successfully inducing regeneration in the spinal cord, and to the merits of this approach for tumor reprogramming

The paper “Brief Local Application of Progesterone via a Wearable Bioreactor Induces Long-Term Regenerative Response in Adult Xenopus Hindlimb” has been published in the journal Cell Reports.

What causes phantom limb — it’s all in the brain

A new brain imaging study could finally explain how and why humans with amputees can still feel their “phantom limbs“. They found that brains maintain incredibly detailed neural maps of the missing limbs even decades after amputation, which could explain the phenomenon.

Image credits Golan Levin / Flickr.

An Oxford University team, comprised of Sanne Kikkert and her colleagues from the Hand and Brain Lab and led by associate professor Tamar Makin, performed a new ultra-high resolution brain imaging study to get to the bottom of the phantom limb.

Almost all people who have lost a limb have some sensation that it is still there, and it’s thought that around 80 percent of amputees experience some level of pain associated with the missing limb. For some the pain is so great it is hugely debilitating. But many phantom limb patients can not only feel the presence of or sensation in the missing limb but also “control” it voluntarily. The participants were asked to move the phantom fingers of an amputated hand while their brains were being scanned so the researchers could map the limb’s representation in the brain.

Previous work has shown that moving an amputated hand corresponds to a spike in brain activity, but we’ve never been able to determine if this is due to some abnormal activity following the amputation, or if it corresponds to cortical territory dedicated to the limb.

Kikkert and her team found the patterns of brain activity have striking similarities to “normal” hand representation. For example, both amputees and the control (two-handed) group showed the same spatial distribution of the fingers in relation to each other. They were able to prove that the hand maps of phantom hands were within the range of those found in the control group. This is quite stunning, considering that the amputee participants lost their hands 25 to 31 years prior.

Brain imaging reveals detailed maps of the individual fingers of the hand in amputees (bottom) that are very similar compared to the hand maps of the two-handed control participants (top).
Image credits Sanne Kikkert et al.

They also showed that the neural activity isn’t a result of muscles or nerves activating in the remaining part of the limb, as was previously thought — these hand maps remained the same in participants amputated above the elbow (so they lacked the muscles) or those with nerve damage. But it’s still unclear if these hand maps cause phantom limb sensations or if the sensations themselves enforce these areas in the brain.

One exciting implication of this study is that its findings go against traditional beliefs of how the body’s sensory map is formed and maintained in the brain. The somatosensory homunculus is a highly organized structure, very striking in that the body parts are laid out in the brain similarly to how they are on the body — and up to now, the prevailing belief was that it required a constant stream of sensory information from the body to maintain its shape.

A diagram of the sensory homunculus, showing how different body parts are mapped in different areas of the brain.
Image via Wikimedia.

In the case of amputation, it was believed that areas of the body near that limb on the homunculus would take over its territory. A 2013 study by Tamar Makin and colleagues showed that, following amputation, the remaining hand starts using the brain territory of the missing hand. Their study also showed that the more participants used their remaining hand to complete daily activities, the more that hand took up the amputated limb’s brain resources, probably to support the overuse of the intact hand.

Kikkert also found signs of reorganisation in her subjects’ missing hand area of the brain, as well as the detailed hand maps. This suggests that following amputation, the original functionality of the area is maintained at the same time as reorganization takes place — something we’ve never been able to determine before.

The team reports their findings “reopen the question of what happens to a cortical territory once its main inputs are removed”. Their work allows us to better understand the homunculus, and could prove invaluable in the further development of neuroprosthetics, complete with tactile sensibility.

The full paper “Revealing the neural fingerprints of a missing hand” was published online in the journal eLife.

Human limbs might have evolved from shark gills

A controversial idea has just received some significant backing, as a group of Cambridge researchers found evidence supporting human limbs evolving from shark gills.

Credit: J. Andrew Gillis

In 1878, German anatomist Karl Gegenbaur proposed an evolutionary link between the gills of cartilaginous fish (such as skates and sharks) and the limbs of vertebrates. The idea was popular for a short bit, but was then generally discarded due to the lack of supporting evidence in the fossil record. However, support may come in the form of a genetic study – specifically, something called the Sonic hedgehog gene.

“Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches,” the study writes.

The Sonic hedgehog gene makes sure all your limbs are in the right place and have the right size. It dictates how the limbs will grow, maintaining the right direction for the skeleton growth. In cartilaginous fish, the gills are protected by flaps of skin supported by arches of cartilage. Interestingly, the purpose of the Sonic hedgehog gene plays the same role for the fish, directing the growth of gills and cartilage. This could indicate that the gene’s function remained unchanged across millions of years of evolution. Writing in this week’s edition of the journal Development, MBL scientist Andrew Gillis and his colleagues support this idea:

“Gegenbaur looked at the way that these branchial rays connect to the gill arches and noticed that it looks very similar to the way that the fin and limb skeleton articulates with the shoulder. The branchial rays extend like a series of fingers down the side of a shark gill arch,” said Andrew Gillis, who led the research, in a statement. “The fact that the Sonic hedgehog gene performs the same two functions in the development of gill arches and branchial rays in skate embryos as it does in the development of limbs in mammal embryos may help explain how Gegenbaur arrived at his controversial theory on the origin of fins and limbs.”

In order to show that the gene works in the same way, they inhibited it at several stages of skate’s development. They found that when inhibited early in development, branchial rays grew on the wrong side of the cartilage arch. When inhibited later in development, the branchial rays grew on the correct side, but were fewer in number.

“Taken to the extreme, these experiments could be interpreted as evidence that limbs share a genetic programme with gill arches because fins and limbs evolved by transformation of a gill arch in an ancestral vertebrate, as proposed by Gegenbaur,” Gillis said.

“However, it could also be that these structures evolved separately, but re-used the same pre-existing genetic programme. Without fossil evidence this remains a bit of a mystery — there is a gap in the fossil record between species with no fins and then suddenly species with paired fins — so we can’t really be sure yet how paired appendages evolved.”

Of course, this is still a hotly debated claim. It doesn’t seem likely for this gene to develop separately, but the fossil evidence is still missing. Additional research is needed to fully compare the functions of the gene, but even if this is further confirmed, I doubt the theory will be widely accepted without fossil evidence. Unfortunately, this type of evidence can be difficult or impossible to find, so this will likely remain an open question for year to come. But the premise is there, and the prospect is certainly interesting.

Journal Reference: A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs

Evolving legs from fins was surprisingly simple, new study finds

New research shows that the first vertebrates had a surprisingly easy time adapting from fins to legs. A new study found that 360 million years ago, legs and fins had no major structural difference.

Axolotls at Vancouver Aquarium. Photo by ZeWrestler.

The research was carried out by Dr Marcello Ruta from the School of Life Sciences at the University of Lincoln and Professor Matthew Wills from the Milner Centre for Evolution at the University of Bath in the UK. They overturned the classical belief that the appearance of legs triggered a diversification in vertebrate skeletons, finding that similar levels of anatomical diversity within their fins and limbs, despite the fact that their skeletons were constructed in very different ways.

The evolution of limbs triggered a revolution in animal evolution, opening up a new realm for animals: dry land. However, they weren’t quick in giving up the fin skeletal structure. Dr Marcello Ruta said:

“Our work investigated how quickly the first legged vertebrates blossomed out to explore new skeletal constructions, with surprising results. We might expect that early tetrapods evolved limbs that were more complex and diverse than the fins of their aquatic predecessors. However, although radically different from limbs, the fins of the distant fish-like forerunners of tetrapods display a remarkable array of subtly varying traits.

“This variation may point to a previously unsuspected range of biomechanical functions in their fins, despite the fact that those ancestors lived exclusively in water.”

Professor Matthew Wills added:

“It has usually been assumed that when organisms evolve novel attributes that enable them to colonise fundamentally new environments — as in the move from water to land — this should trigger rapid evolutionary diversification and be accompanied by an increase in structural variety. Our work challenges this received wisdom, and shows that, at least in the case of the evolution of early tetrapods, key innovations did not quickly lead to greater anatomical variety.

“For the first time, legs had evolved to fulfill new functions. Not only must they be able to support the weight of the body on land, but they also needed to enable the animal to walk. Perhaps these dual requirements limited the number of ways in which these first legs could function and evolve, thereby constraining their range of variability.”

The study has significant implications for how biological systems are studied – both past and present – especially when we’re dealing with diversification stages. It also shows that no matter how significant and vital the evolutionary stage was, it can still take millions of years to pan out.

Journal Reference:

  1. Marcello Ruta, Matthew A. Wills. Comparable disparity in the appendicular skeleton across the fish-tetrapod transition, and the morphological gap between fish and tetrapod postcrania.Palaeontology, 2016; 59 (2): 249 DOI: 10.1111/pala.12227

Stem Cell therapy could help us grow back fingers

Mammals can naturally regenerate the very top of their fingers and toes after amputation; starting from this idea, researchers have demonstrated the mechanism that describes this process, and explain how stem cells from nails could play a pivotal role in future regeneration of entire fingers.

fingers

A study conducted on mice showed that the chemical signal that triggers stem cells to develop into new nail tissue also attracts nerves that promote bone and nerve regeneration. The study suggests that nail stem cells could be used to develop new regeneration treatments for amputees.

Mice are pretty similar to humans in thhis regard – in both species, regenerating a finger starts with regenerating the nail. But whether the amputated portion of the digit actually takes place depends on exactly where the amputation occurs – if the stem cells in the nails are amputated as well, then no regeneration takes place. But if a small portion of the nail still remains in place, then it does regenerate. To understand exactly why these stem cells are so important, researchers turned to mice.

The two unfortunate groups of mice were separated into two groups – one control group, and one which was treated with a drug that made them unable to make the signals for new nail cells to develop. The second group was unable to regenerate, while the first one did this just fine, in time. When the signal was replenished, the second group resumed regeneration.

Limb regeneration is a very interesting field for biologists at the moment; a vast number of animals can regenerate lost limbs, most notably amphibians – aquatic salamanders can regenerate complete limbs, and even parts of their heart, by a process which involves their immune system. By studying species which are close to us and understanding the mechanism through which they regenerate, we could some day apply the same techniques to humans.

Via Discovery