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 regenerative potential after hindlimb amputation in adult Xenopus. In addition, Prog can regulate bioelectric cell state via changes in ion flux ( Luoma et al., 2012 Patrat et al., 2002), and bioelectric signaling has been proved to drive regeneration and patterning in multiple assays in Xenopus and other model systems ( Herrera-Rincon and Levin, 2016 Levin, 2011, 2014 Lobikin et al., 2015 McLaughlin and Levin, 2018 Tseng and Levin, 2013 Tseng et al., 2007, 2010). Progesterone (Prog) is a potent neurosteroid that showed promise for peripheral nerve regeneration (promoting nerve repair) ( Koenig et al., 2000) and bridging in severed rabbit facial nerves ( ChГЎvez-Delgado et al., 2003), canine sciatic nerves ( Rosales-Cortes et al., 2003), and non-neural tissue remodeling by modulating the inflammatory response to drive wound healing, angiogenesis ( McEwen, 1991 Routley and Ashcroft, 2009), and bone remodeling ( Hennighausen and Robinson, 2001). We recently showed that a wearable bioreactor ( Hechavarria et al., 2010) positively affects regenerative capabilities ( Golding et al., 2016). We thus sought to develop a methodology to test targeted delivery of small-molecule electroceuticals in adult animals to overcome their lack of regenerative response with an early, local, and brief intervention. We have previously shown the induction of tail regeneration in non-regenerative Xenopus stages by targeting the bioelectric state with small-molecule drugs ( Adams et al., 2007, 2013 Tseng et al., 2010) however, those studies involved juvenile frogs and used whole-animal exposure to inductive cocktails, a strategy that is limiting because of the extensive use of bioelectric signaling throughout the body. However, this capability declines markedly throughout development, and in later tadpole and post-metamorphic animals, the resultant regenerate is a hypomorphic cartilage вЂвЂspikeвЂ™вЂ™ ( Dent, 1962). Xenopus laevis exhibits a regeneration response similar to that of urodele axolotls, which can reform a functional limb after amputation. Many animals possess a remarkable capacity for appendage regeneration, which is not found in humans despite a high degree of gene homology ( Brockes and Kumar, 2008). Despite concerted efforts on the mechanisms of regeneration ( Kawakami et al., 2006 Leppik et al., 2015), there are few reports of rebuilding and/or repairing damaged or lost limbs in non-regenerative animals, and there is a pressing need for a tractable vertebrate model in which to test potential interventions. An ideal solution would trigger the native limb-building pathways in vivo, to restore a functional limb from the patientвЂ™s own cells. The repair of damaged limbs and organs in humans is of paramount importance, as 2 million Americans are living with limb amputations ( Ziegler-Graham et al., 2008). 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. 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. 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. 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.

The induction of limb repair in adult vertebrates is a pressing, unsolved problem.

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