For millions of patients who have lost limbs, the possibility of regaining function through natural regeneration remains out of reach. Regrowth of legs and arms remains the province of animals such as salamanders, or perhaps superheroes. But now, scientists at Tufts University and Harvard University’s Wyss Institute report on their use of a novel multidrug treatment (MDT), delivered through a wearable device, which allowed adult African clawed frogs (Xenopus laevis)—which can’t naturally regenerate limbs—to regrow amputated hind legs. The regrown limbs enabled the animals to move around in a manner similar to normal frogs, and even responded to touch.
While most studies on limb regeneration have focused on animals such as the axolotle, which demonstrates natural regrowth capabilities, the newly reported study in frogs could help direct future research towards limb regeneration in mammals, including humans. “It’s exciting to see that the drugs we selected were helping to create an almost complete limb,” said Nirosha Murugan, PhD, research affiliate at the Allen Discovery Center at Tufts and first author of the team’s published paper in Science Advances. “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.”
Murugan and colleagues describe their achievement in a paper titled, “Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis,” in which they concluded, “This provides a proof of principle for kickstarting complex organ regeneration responses in vertebrate models using compound interventions consisting of a drug blend and a wearable bioreactor delivery device.”
The prevalence of human limb loss in the United States alone is expected to increase substantially over the next 30 years, potentially affecting 3.6 million people per year by 2050, the authors wrote. This will leave individuals with diabetes, war veterans, survivors of trauma, and those suffering from peripheral artery disease with limited options following an amputation. “Despite significant technological advances, clinicians still lack tools to facilitate the recovery or reversal of tissue loss, while prosthetics offer only limited functional restoration of the patient’s own limbs,” the team continued. “Thus, identifying and potentiating the underlying programs that drive limb regrowth would be of great interest to medical and research fields.”
Many creatures—including salamanders, starfish, crabs, and lizards—have the capability of full regeneration of at least some limbs. Flatworms can even be cut up into pieces, with each piece reconstructing an entire organism. Humans are capable of closing wounds with new tissue growth, and our livers have a remarkable, almost flatworm-like capability of regenerating to full size after a 50% loss. However, loss of a large and structurally complex limb—an arm or leg—can not be restored by any natural process of regeneration in humans or other mammals. In fact, we tend to cover major injuries with an amorphous mass of scar tissue, protecting it from further blood loss and infection and preventing further growth.
X. laevis is a common model organism that has previously been used to address questions in developmental biology and regenerative medicine, the authors noted. “Organisms such as X. laevis—whose limited regenerative capacities in adulthood mirror those of humans—are important models with which to test interventions that can restore form and function.” Previous work by the Tufts team showed a significant degree of Xenopus limb growth could be triggered by a single drug, progesterone. However, the resulting limb grew as a spike and was far from the more normally shaped, functional limb. “Our recent work in adult Xenopus showed a significant degree of outgrowth induced by progesterone,” they wrote. “However, the resulting regenerate was far from fully functional and repatterned limbs were not achieved.”
For their newly reported study in adult frogs, the Tufts researchers triggered the regenerative process by enclosing the wound in a silicone cap, called a BioDome, which contained a silk protein gel loaded with a five-drug cocktail. “We hypothesized that kickstarting a limb-building routine required two components acting at a very early initiating stage of the process: first, an enclosed and permissive microenvironment that enables the wound cells to control the biochemical milieu following injury and, second, an instructive set of signals that specifically trigger a limb-building program.” The multidrug treatment cocktail consisted of five small-molecule compounds (BDNF, GH, 1,4-DPCA, RD5, and RA), which, in previous studies had been independently shown to have marked pro-regenerative effects.
The BioDome device essentially sealed the drug cocktail over the stump for just 24 hours, and that brief treatment was enough to set in motion an 18-month period of regrowth that restored a functional leg. “Treated animals displayed a marked delay of wound closure, followed by long-term (18-month) growth outcomes including increased bone length, soft tissue patterning, and neuromuscular repair,” the investigators wrote.
Each drug fulfilled a different purpose, including tamping down inflammation, inhibiting the production of collagen which would lead to scarring, and encouraging the new growth of nerve fibers, blood vessels, and muscle. The combination of the multidrug cocktail and bioreactor provided a local environment and signals that tipped the scales away from the natural tendency to close off the stump, and toward the regenerative process.
The researchers observed dramatic growth of tissue in many of the treated frogs, recreating an almost fully functional leg. The new limbs demonstrated bone structure that extended with features similar to a natural limb’s bone structure, a richer complement of internal tissues (including neurons), and several “toes” grew from the end of the limb, although without the support of underlying bone. “Histologically, the regenerating limbs contained nerves, smooth muscle displaying integration of blood vessels, and reorganization of the extracellular matrix (ECM) proteins involved in tissue remodeling,” the scientists continued.
Importantly, the regrown limb moved and responded to stimuli such as a touch from a stiff fiber, “… indicating that they had regained significant reinnervation and neuromuscular reintegration compared to normal functionality preinjury …” and the frogs were able to make use of the regrown limb for swimming through water, moving much like a normal frog would. “Most notably, the animals used the newly formed limb to ambulate in a manner similar to that of wild-type frogs,” the scientists stated.“It’s exciting to see that the drugs we selected were helping to create an almost complete limb,” said Murugan. “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 researchers explored the mechanisms by which the brief intervention could lead to long-term growth. Within the first few days after treatment, they detected the activation of known molecular pathways that are normally used in a developing embryo to help the body take shape. Activation of these pathways could allow the burden of growth and organization of tissue to be handled by the limb itself, similar to how it occurs in an embryo, rather than require ongoing therapeutic intervention over the many months it takes to grow the limb.
Animals naturally capable of regeneration live mostly in an aquatic environment. The first stage of growth after loss of a limb is the formation of a mass of stem cells at the end of the stump called a blastema, which is used to gradually reconstruct the lost body part. The wound is rapidly covered by skin cells within the first 24 hours after the injury, protecting the reconstructing tissue underneath.
“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, PhD, the 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.”
The five-drug cocktail represents a significant milestone toward the restoration of fully functional frog limbs and suggests further exploration of drug and growth factor combinations could lead to regrowth of limbs that are even more functionally complete, with normal digits, webbing, and more detailed skeletal and muscular features.
“We’ll be testing how this treatment could apply to mammals next,” said corresponding author Michael Levin, PhD, the Vannevar Bush professor of biology in the School of Arts & Sciences, director of the Allen Discovery Center at Tufts, and associate faculty member of the Wyss Institute.
“Covering the open wound with a liquid environment under the BioDome, with the right drug cocktail, could provide the necessary first signals to set the regenerative process in motion,” he said. “It’s a strategy focused on triggering dormant, inherent anatomical patterning programs, not micromanaging complex growth, since adult animals still have the information needed to make their body structures.”
The authors concluded, “The generalizability of this specific MDT must next be tested in mammals. We suggest that the overall strategy of providing wound cells with an aqueous, amniotic-like environment, which is uniquely given through our bioreactor, that contains pro-regenerative signals is likely to be an effective method for kickstarting biomedically relevant growth and patterning cascades that are too complex to directly implement.”