Scientists at Tufts University have developed a wearable bioreactor that can promote partial regeneration of hindlimbs in adult African clawed frogs (Xenopus laevis) following amputation. Tests showed that the hydrogel-filled device, which contains silk proteins and progesterone, induced the development of a rudimentary paddle-like limb that was more structured than the typical cartilaginous spike that regenerates in untreated frogs. Compared with untreated amputees, the bioreactor-treated animals were also more active, and capable of swimming more like normal frogs.

The researchers say their experiments, described in Cell Reports, will represent a model for testing “therapeutic cocktails” in vertebrate hindlimb regeneration. “At best, adult frogs normally grow back only a featureless, thin, cartilaginous spike,” said senior author Michael Levin, Ph.D., a developmental biologist at the Allen Discovery Center at Tufts University. “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 team’s paper is titled, “Brief local application of progesterone via a wearable bioreactor induces long-term regenerative response in adult Xenopus hindlimb.”

Approximately two million people in the U.S. have a limb amputation, and while an ideal solution would involve the ability to trigger the body to set in motion the relevant pathways for restoring limb structure using its own cells, there have been few reports of success in rebuilding or repairing damaged or lost limbs in animals that don’t normally regenerate such structures, the authors write. Scientists also don’t yet have “a tractable vertebrate model in which to test potential interventions.” Although many animals can regenerate amputated appendages, this capacity in adult Xenopus laevis frogs is restricted to the formation of a cartilage “spike.”

The Tufts University team had previously developed a wearable bioreactor that could positively impact on regenerative capabilities. For their reported studies the team combined the bioreactor system with progesterone (Prog), a neurosteroid that previous studies have found may help to promote peripheral nerve repair and support non-neural tissue remodeling by modulating inflammatory responses to support wound healing, angiogenesis, and bone remodeling. “Due to its potent and broad actions on neural and non-neural tissue remodeling, as well as its ability to influence bioelectric signaling, we asked whether treatment with a Prog-containing silk device immediately after amputation would improve cellular dynamics and regeneration potential after hindlimb amputation in adult Xenopus.”

The team 3D printed the bioreactor from silicon and filled it with a hydrogel containing hydrating silk proteins to promote healing and regeneration, and progesterone. They tested the bioreactor on groups of experimental, sham, and control adult Xenopus frogs. For the experimental and sham groups, the bioreactor (containing progesterone for the experimental group) was sutured onto the site of amputation immediately after limb amputation, but removed after 24 hours.

Initial tests confirmed that the bioreactor released progesterone to the site of injury. Early in the regenerative process frogs in the experimental group also exhibited reduced immune infiltration, with evidence that bioreactor treatment induced scar-free wound healing. Treated animals also showed signs of regenerating nerves, “which are key markers of specialized tissue organization,” the authors write.

Over the course of 9.5 months, the animals that had been treated using the progesterone bioreactor exhibited a greater degree of limb regeneration than the sham and control groups. The bioreactor-progesterone treatment prompted the formation of a paddle-like limb assembly that more closely resembled a fully formed limb than the characteristic spike formed by unaided regeneration. Macroscopic analysis indicated that the 24-hour treatment triggered long-term reorganization of both soft tissue and bone, with patterning that is “consistent with joint formation,” the team wrote.

“The bioreactor device created a supportive environment for the wound where the tissue could grow as it did during embryogenesis,” said Dr. Levin. “A very brief application of bioreactor and its payload triggered months of tissue growth and patterning.” The treated animals were also far more active than the untreated animals, and the regenerated paddle-like limbs could be used in swimming and active behaviors that were similar to a native limb.

Interestingly, RNA sequencing studies indicated that progesterone treatment using the bioreactor altered gene expression and cell processes and modified transcriptional responses. “Significant cell processes exclusively upregulated after Prog-device treatment included a list of key genes that are involved in redox stress, serotonergic transmission, or leukocyte proliferation,” the team noted, “while important downregulated processes included genes responsible for neurotransmission signaling and dynamic changes in Ca2+and K+… Thus, our analysis reveals profound transcriptional remodeling of the regeneration blastema in Xenopus induced by a Prog-containing bioreactor and suggests numerous pathway targets for subsequent study and therapeutic interventions.”

Downregulated scarring and immune responses in the experimental group, were indicative that the progesterone had dampened the body’s natural reaction to injury, to help allow regeneration to progress. “In both reproduction and its newly discovered role in brain functioning, progesterone’s actions are local or tissue-specific,” said first author Celia Herrera-Rincon, Ph.D., a neuroscientist in Dr. Levin’s laboratory. “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 authors claim that their studies provide proof-of-principal that brief use of an integrated device for delivery of drugs represents a viable strategy for inducing and maintaining long-term regenerative responses. They acknowledge that what still isn’t known is what degree of micromanagement of the regenerative process will be needed besides providing the initial signal that flicks the regenerative switch. “Our data establish a platform to test ‘master regulator’ therapeutics, in which a very brief treatment ‘kick-starts’ a long program of growth and remodeling,” they conclude. “These findings reveal that the adult Xenopus limb is capable of considerable growth and morphogenesis and illustrates a roadmap for interventions that can be used to probe and improve the mechanisms of complex appendage regeneration in vertebrate models.” The authors suggest that a successful strategy may well hinge around a process of “guided self-assembly” that involves occasional external manipulation of endogenous morphogenic processes to keep growth on course.

The team hopes to replicate their Xenopus results in mammals. Prior studies have indicated that mice can partially regenerate amputated fingertips, but with some hindrances. “Almost all good regenerators are aquatic,” explained Dr. Levin. “You can imagine why this matters: 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.”