Scientists from Tufts University have developed a portable bioreactor that partially regenerates hind paws in adult African clawed frogs (Xenopus laevis) after amputation. Tests showed that the hydrogel-filled device, containing silk proteins and progesterone, induced the development of a rudimentary paddle-like limb that was more structured than the typical cartilaginous peak that regenerates in untreated frogs. Compared to untreated amputees, animals treated with bioreactor were also more active and able to swim more like normal frogs.
The researchers say their experiments, described in Cell reports, will propose a model for testing "therapeutic cocktails" in the regeneration of the backwind through vertebrates. "At best, adult frogs normally only regain a cartilaginous, thin, cartilaginous peak," said senior author Michael Levin, Ph.D., a developmental biologist at the Allen Discovery Center at Tufts University. "Our procedure caused a regenerative response that they normally never have, which resulted in larger, more structured appendages, and the bioreactor device caused very complex downstream outcomes that bioengines can not yet micromanage directly." The team report is titled: " Short local application of progesterone via a portable bioreactor induces long-term regenerative response in adults Xenopus hind leg. "
Approximately two million people in the US have an amputation of the limbs, and although an ideal solution would include the ability to urge the body to activate the relevant pathways to restore the limb structure using its own cells, its there have been few reports of success in reconstruction or repairing damaged or lost limbs in animals that do not normally regenerate such structures, the authors write. Scientists do not yet have "a traceable vertebrate model to test potential interventions." Although many animals can regenerate amputated appendages, this capacity is in adults Xenopus laevis frogs are limited to the formation of a cartilage "spike".
The Tufts University team had previously developed a portable bioreactor that could have a positive effect on the regenerative possibilities. For their reported studies, the team combined the bioreactor system with progesterone (Prog), a neurosteroid whose previous studies have shown that it can promote peripheral nerve repair and non-neural tissue remodeling by modulating inflammatory responses to support wound healing, angiogenesis and bone remodeling . "Because of the powerful and broad actions on the remodeling of neural and non-neural tissues, as well as their ability to influence bioelectric signaling, we asked whether treatment with a Prog-containing silk system immediately after amputation cellular dynamics and regeneration potential after the hindleg would improve amputation in adult Xenopus. "
The 3D team pushed the silicon bioreactor and filled it with a hydrogel that contained hydrating silk proteins to promote healing and regeneration and progesterone. They tested the bioreactor on groups of experimental, apparent and control adults Xenopus frogs. For the experimental and sham groups, the bioreactor (containing progesterone for the experimental group) was sutured at the site of amputation immediately after limb amputation, but after 24 hours.
Initial tests confirmed that the bioreactor progesterone expired at the site of the injury. Early in the regenerative process, frogs in the experimental group also showed reduced immunological infiltration, with evidence that bioreactor treatment induced scar free wound healing. Treated animals also showed signs of regenerating nerves, "which are the main features of specialized tissue organization," the authors write.
In the course of 9.5 months, the animals treated with the progesterone bioreactor showed a greater degree of limb regeneration than the sham and control groups. The bioreactor-progesterone treatment led to the formation of a paddle-like limb assembly that more closely resembled a fully formed leg than the typical spike formed by the autonomous regeneration. Macroscopic analysis indicated that the 24-hour treatment caused a 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 short application of the bioreactor and its payload caused months of tissue growth and pattern formation." The treated animals were also much more active than the untreated animals, and the regenerated paddle-like limbs could be used in swimming and active behavior similar to a natural limb.
Interestingly, RNA sequencing studies indicated that progesterone treatment using the bioreactor changed gene expression and cell processes and modified transcriptional responses. "Significant cellular processes that have been exclusively upregulated after treatment with Prog devices include a list of key genes involved in redox stress, serotonergic transmission or leukocyte proliferation," the team noted, "while major downregulated processes include genes responsible for signaling by neurotransmission. and dynamic changes in Ca2+and K+… Thus our analysis reveals a profound transcriptional remodeling of the regeneration blastema Xenopus induced by a Prog-containing bioreactor and suggests numerous pathway targets for subsequent studies and therapeutic interventions. "
Downregulated scars and immune responses in the experimental group, were indicative that the progesterone had attenuated the body's natural response to injury, to help allow regeneration to progress. "In both the reproduction and the newly discovered role in the functioning of the brain, the actions of progesterone are local or tissue-specific," said the first author Celia Herrera-Rincon, Ph.D., a neuroscientist in the Dr. Levin. "What we are demonstrating with this approach is that reproduction, brain processing and regeneration may be closer than we think, perhaps sharing paths and elements of a common – and so far not fully understood – bioelectric code."
The authors claim that their studies provide evidence that the short use of an integrated drug delivery device represents a viable strategy for the generation and maintenance of long-term regenerative responses. They recognize that what is still not known, the degree of micromanagement of the regenerative process will be needed in addition to providing the initial signal that vibrates the regenerative switch. "Our data forms a platform for testing" master-regulator "therapies, where a very short treatment a long program of growth and remodeling" kick-start & # 39; ", they conclude. "These findings reveal that the adult Xenopus limb is capable of significant growth and morphogenesis and illustrates a roadmap for interventions that can be used to investigate and improve the mechanisms of complex appendage regeneration in vertebrate models. "The authors suggest that a successful strategy can indeed pivot around a process of" guided self-study "-assembly" in which occasional external manipulation of endogenous morphogenetic processes takes place in order to keep growth going.
The team hopes to replicate them Xenopus results in mammals. Previous studies have shown that mice can regenerate partially amputated fingertips, but with some obstacles. "Almost all good regenerators are in the water," Dr. Levin out. "You can imagine why this is important: a mouse that loses a finger or hand and then grinds the fine regenerative cells in the floor covering material while walking around, it is unlikely to experience significant regeneration of the limbs."