Brief multi‐drug treatment via a wearable silk‐hydrogel bioreactor induces long‐term limb re‐patterning and regeneration in adult Xenopus Laevis

Abstract

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.