Limb regeneration is a frontier in biomedical science. Identifying triggers of innate morphogenetic responses in vivo to induce the growth of healthy patterned tissue would address the needs of millions of patients, from diabetics to victims of trauma. Organisms such as Xenopus 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. Here, we demonstrate long-term (18 months) regrowth, marked tissue repatterning, and functional restoration of an amputated X. laevis hindlimb following a 24-hour exposure to a multidrug, pro-regenerative treatment delivered by a wearable bioreactor. Regenerated tissues composed of skin, bone, vasculature, and nerves significantly exceeded the complexity and sensorimotor capacities of untreated and control animals’ hypomorphic spikes. RNA sequencing of early tissue buds revealed activation of developmental pathways such as Wnt/β-catenin, TGF-β, hedgehog, and Notch. These data demonstrate the successful “kickstarting” of endogenous regenerative pathways in a vertebrate model.
In this study, we examined how time perception, a psychological factor, impacts the physiological response to prolonged, voluntary breath holding. Participants (n = 26) held their breath while watching a distorted timer that made it appear as though time was moving up to 40% faster or slower than real time. We monitored total breath‐holding duration under different time manipulation conditions as well as the onset of involuntary breathing movements. This physiological breaking point marks the end of the “easy‐going” phase of apnea and the start of the “struggle” phase. Based on prior work showing that psychological factors, such as attention and motivation, can influence the length of the struggle phase, we hypothesized that manipulating the perception of time would affect overall breath‐holding duration by changing the duration of the struggle phase, but not the easy‐going phase. We found that time perception can be successfully manipulated using a distorted timekeeper, and total breath‐holding duration correlated with perceived time, not actual time. Contrary to our hypothesis, this effect was attributable to changes in the onset of the physiological breaking point, not changes in the length of the struggle phase. These results demonstrate that unconscious psychological factors and cognitive processes can significantly influence fundamental physiological processes.
Limb regeneration requires the temporal and spatial patterning of molecular signals within a suitable microenvironment such that differentiation and growth can be orchestrated. Despite advances in limb prostheses, ideal solutions to limb loss will involve re‐structuring native tissues to regain biological function. Our novel biomedical engineering approach was designed to induce patterning by controlling the microenvironment at the wound site in amputated hindlimbs of Xenopus laevis, without having to micromanage every molecular cascade. Post‐metamorphic, non‐regenerative frogs which had undergone hindlimb amputation were treated with an innovative wearable bioreactor composed of a silk‐hydrogel infused with 5 pro‐regenerative compounds for only 24 hours. Animals were maintained for 18 months where morphometric and histological analyses were performed at regular intervals to monitor neural, vascular, and osseous repatterning. The 24‐hr. brief exposure to the bioreactor induced remarkable growth with patterned structures not observed in untreated controls. Bone remodelling, re‐vascularization, and digit patterning were observed 18 months after the initial 24 hour bioreactor treatment. 18‐month post treatment regenerates also regained sensorimotor abilities within the regenerated hindlimb. Whole transcriptomic analysis at early regenerative periods confirmed changes to blastemal profiles related only to drug‐reactor exposures, which included changes in differential gene expression profiles related to crucial pro‐regenerative mechanisms such as sox2, KLf4, WNT pathway genes and upregulation of pro‐regenerative inflammatory and neural pathways. Our data provide strong evidence that a non‐regenerative animal can be induced to re‐pattern limbs when exposed to a controlled bioreactor device. This first induction of an advanced limb regenerative response in fully adult organisms suggests using a biomedical engineering approach with silk‐hydrogel as a bioreactor for simultaneous and compound delivery of multiple drug compounds is a promising avenue for induction of regeneration in adult, non‐ regenerative species.
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