A team of scientists designed a device that can induce partial hindlimb regeneration in adult aquatic African clawed frogs (Xenopus laevis) by “kick-starting” tissue repair at the amputation site. They introduce a new model for testing “electroceuticals,” or cell-stimulating therapies.
“At best, adult frogs normally grow back only a featureless, thin, cartilaginous spike,” says senior author Michael Levin (@drmichaellevin), 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 scientists 3D printed the bioreactor out of silicon and filled it with hydrogel–a sticky glob of polymers. They laced the hydrogel with hydrating silk proteins that promote healing and regeneration, then added progesterone. Progesterone is best known for its role in preparing the uterus for pregnancy, but the hormone has also been shown to promote nerve, blood vessel, and bone tissue repair.
The researchers split the frogs into three groups: experimental, control, and sham. For the experimental and sham group, they sutured the device on the frogs immediately after limb amputation. In the experimental group, the bioreactor released progesterone onto the amputation site. In all cases, they removed the devices after 24 hours.
The repair of damaged limbs and organs in humans is of paramount importance, as ∼2 million Americans are living with limb amputations.
The treatment described herein induced large, paddle-shaped regenerates, demonstrating a clear improvement over the well-known cartilaginous spike. The treatment appears to reactivate the early regenerative response, because transcriptional networks are specifically suppressed or enriched as soon as 24 hr after treatment, and morphological differences are already visible at 2 weeks post-amputation. We previously demonstrated that that body-wide pharmacological modulation of Vmem can induce functional regeneration of the froglet leg at a non-regenerative stage. To achieve localized delivery (essential for broadening the class of treatments that can be used without toxicity), we designed a wearable bioengineered bioreactor: the wearable bioreactor. The silk-hydrogel-based device, even though present very briefly, created a pro-regenerative environment, enhancing bone remodeling. Micro-computed tomography (CT) quantifications revealed that animals with devices had a significant increase in bone volume, surface area, and density. Qualitative observations on immunostained sections also detected the presence of nerve tissue or SMA+ vessels in treated animals. Although morphologically regenerates from treated animals tended to be longer than those from no-device animals, no pattern or complex structures or shapes were detected, which differs from the rod-shaped spike. Our data suggest the brief application of a Prog-containing bioreactor as a promising modality for initiating complex, long-lasting, pro-regenerative outcomes.
Highlights – Wearable Bioreactor Induces Long-Term Regenerative Response in Adult Xenopus Hindlimb
• Adult Xenopus laevis frogs are capable of induced increased regenerative response
• Improved limb regeneration is driven by a wearable bioreactor containing progesterone
• Improvements occur at molecular, anatomical, and behavioral (functional) levels
• A 24-hr treatment is sufficient to trigger many months of regenerative growth
Abstract – Wearable Bioreactor Induces Long-Term Regenerative Response in Adult Xenopus Hindlimb
The induction of limb repair in adult vertebrates is a pressing, unsolved problem. Here, we characterize the effects of an integrated device that delivers drugs to severed hindlimbs of adult Xenopus laevis, which normally regenerate cartilaginous spikes after amputation. A wearable bioreactor containing a silk protein-based hydrogel that delivered progesterone to the wound site immediately after hindlimb amputation for only 24 hr induced the regeneration of paddle-like structures in adult frogs. Molecular markers, morphometric analysis, X-ray imaging, immunofluorescence, and behavioral assays were used to characterize the differences between the paddle-like structures of successful regenerates and hypomorphic spikes that grew in untreated animals. Our experiments establish a model for testing therapeutic cocktails in vertebrate hindlimb regeneration, identify pro-regenerative activities of progesterone-containing bioreactors, and provide proof of principle of brief use of integrated device-based delivery of small-molecule drugs as a viable strategy to induce and maintain a long-term regenerative response.
Previous Tufts work found bioelectric mirroring on the uninjured leg opposed to the amputated leg
Tufts University biologists have discovered that amputation of one limb is immediately reflected in the bioelectric properties of the contralateral, or opposing, the un-damaged limb of developing frogs. The pattern of bioelectric depolarization in the uninjured leg is directly correlated to the position and type of injury, indicating that information about damage to tissues is available to their symmetrical counterparts within about 30 seconds of injury.
When they amputated the limbs of froglets still in regeneration stage, the dye revealed a remarkable phenomenon: The un-injured leg exhibited bioelectric states that mirrored the location and type of injury occurring on the opposite side, and the effect was immediate, occurring within 5 seconds.
“What was amazing about this result was that not only did the depolarization in the un-injured leg detect the presence of injury on the other side, it also reflected information about the position of the cut,” said Levin.