Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis

Abstract

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.

Fig. 1.. Twenty-four–hour multidrug treatment delivered by bioreactor improves repatterning in regenerates.

(A) Xenopus hindlimbs were amputated and inserted into silk-based hydrogel devices (yellow arrow) for 24 hours. Hydrogels contained a multidrug treatment (MDT) or no added factors (BD). Some animals received no device (ND). (B) Regenerated MDT hindlimbs were longer than the BD and ND groups by 2.5 mpa, as indicated by growth beyond resection site (red dashed line). At 4 mpa, vascularized structures developed at the distal extension of MDT, but not BD or ND regenerates. At 9 mpa, digit-like projections appeared (blue arrow), contrasting the hypomorphic spikes of BD and ND regenerates (pink arrows). (C) Soft tissues of MDT animals were consistently longer than BD or ND from 8 mpa [F(2,19) = 61.9, P < 0.05]. (D) Micro-CT reconstructions revealed increases in regenerate bone length (yellow arrow) and volume, as well as diffuse bone (purple arrow). (E) Volumetric quantification confirmed increased growth in MDT regenerates [F(2,15) = 11.15, P < 0.001]. (F) Tuberculae complexity increased in MDT-exposed tissue. (G) Micro-CT reconstructions show reestablishment of fine bone structures in MDT regenerates (purple arrow). (H) Longitudinal x-ray imaging indicates gradual accelerated bone growth in MDT [F(2,19) = 31.04, P < 0.05]. Means and SD are presented. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.. MDT-exposed hindlimbs displayed wound closure delays and increased stemness markers, resulting in longer regenerates following amputation.

(A) Wound diameter (green dashed circle) 2 weeks after amputation in MDT animals as evidenced by both sagittal and frontal views. The yellow arrows indicate surgical margins, (B) revealing increased growth associated with MDT-exposed animals relative to ND animals. No significant differences were apparent when comparing BD-exposed and ND animals. ns, not significant. (C and D) SOX2 expression (green), which is a marker of stemness, was greatly increased in blastema sections of the MDT-exposed animals, while little to no expression was observed in another group (counterstained with DAPI, blue). (E) Increased soft tissue growth was also observed 2.5 mpa in the MDT-exposed group, which mirrored the (F) increased length of bone tissues as confirmed by x-ray imaging. Scale bars, 100 μm (A, C, and E). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.. Transcriptomic analysis of early wound tissue reveals gene expression changes associated with regenerative outcomes.

(A) Tissue was collected from the amputation site from MDT and ND animals at 11 hpa, 24 hpa, and 7 dpa and subjected to RNA-seq analysis. (B) Heatmap comparing gene expression levels of MDT animals compared to ND shows significant differential gene expression at 11 hpa that persists to 24 hpa (see color-coded legend). At 7 dpa, however, dynamic gene expression levels return to normal, indicating a period of dynamic gene expression within 24 hours of amputation. Next, we compared the top 20 enriched (C) up-regulated and (D) down-regulated KEGG pathways in X. laevis treated with MDT versus ND overtime. (E and F) Gene set enrichment analysis (GSEA) revealed that inflammatory signaling and cell morphogenesis gene sets were up-regulated in blastema treated with CT at 11 hpa (left) and 24 hpa (middle) (G), which switched to more cellular regulation and metabolic mechanisms at 7 dpa (right). (H to J) We identified a handful of genes that were highly regulated in MDT blastema at 11 hpa (left), 24 hpa (middle), and 7 dpa (right)—particularly with MDT animals as compared to ND animals. Up-regulated genes included immune system–related transcripts (e.g., PTGS2, TGFB1, IL1B, and FOXP3), which suggest an important role of inflammation in pro-regenerative states.

Fig. 4.. Transcriptomic changes reflect regeneration-focused signaling mechanisms.

(A) GSEA enrichment plots displayed major signaling pathways involved in cell morphogenesis that were up-regulated in the early blastema in MDT-exposed animals relative to ND. Specifically, up-regulated genes enriched within each KEGG term are shown within each GSEA plot. FDR (adjusted P value), enrichment score (ES), and normalized ES (NES) are presented with each plot. Cell morphogenesis–related signaling peaks at 11 hours and decreases over time, with WNT, hedgehog, and TGF-β displaying no significant differences in differential gene expression between MDT and ND 7 dpa. (B) Volcano plots depict statistical significance (FDR; adjusted P value) versus magnitude of change (fold change) for rapid visual identification of genes. Data points, representing individual genes, highlight fold change values that are also statistically significant. Statistical significance is measured by −log₁₀(FDR). The fold change was measured by logFC [cutoff at logFC (>0.05)]. Significant GO pathways comparing MDT and ND groups 11 hours, 24 hours, and 7 days comparing MDT versus ND are displayed. Biologically significant results include KIT and IL10 for inflammation (INFL), FGD for fibroblast (FIBR), which varies as a function of time, and SALL4 for appendage morphogenesis (MORPH), which is up-regulated and typically observed during scar-free wound healing in axolotls.

Fig. 5.. GSEA revealed four distinct expression modules indicative of complex tissue repatterning.

CEMiTool was used to identify covarying gene sets as a function of high fold changes in the MDT (CT) and ND animals, yielding modules M1 to M4. (A) Relative expression of modules between time points and conditions (dark red circles = up-regulation; dark blue circles = down-regulation). (B) M2 to M4 were up-regulated in MDT animals versus ND across all time points, peaking at 24 hpa; however, M1 was down-regulated in the MDT condition, most highly at 11 hpa, and decreasing to 7 dpa. M3 genes were up-regulated in MDT relative to ND including acetylcholine activity and muscle activation. M4 genes were up-regulated in MDT relative to ND including glucose metabolism. (C) GO data within the ECM development pathway indicated up-regulation of ADAM family genes at 11 and 24 hpa; however, they were down-regulated at 7 dpa. (D and E) Cross-sectioned regenerates (18 mpa) were double fluorescent–stained for markers of vascularization: smooth muscle actin (red) and basement membrane (green). SMA/laminin⁺ bundles were increased in MDT animals relative to device only or ND groups (P < 0.001). (F) Fibronectin⁺ cartilaginous cores of regenerates displayed increased structural complexity associated with the MDT group relative to all other groups (*P < 0.05). Scale bars, 1 mm. Means and SD are presented.

Fig. 6.. Nerve regeneration and sensorimotor reintegration in MDT-exposed animals are associated with gain of function in regenerated hindlimbs.

(A) Distal (left column) and proximal (right column) sections of the regenerates were labeled with antibodies for an axonal nerve marker, AAT (red), and counterstained with DAPI (blue). Sections obtained from regenerates exposed to the MDT immediately after amputation displayed significantly more AAT⁺ bundles (B) [N = 10 MDT, n = 8 BD, n = 6 ND, Kruskal-Wallis H(3,24) = 15.12, P < 0.005] with wider diameter (C) [Mann-Whitney U(3,24) = 11.74, P = 0.0028] than those sections obtained from ND or BD-exposed animals. (D) Sensorimotor reintegration as assessed by VF filaments applied to the distal tip of the regenerate showed gain of function in the MDT group similar to that of unamputated (wild-type) limbs from ND animals. (E) MDT animals displayed a reliable restoration of sensorimotor function as evidenced by a stimulation force threshold (0g) matching uninjured (UI) animals (P > 0.05). BD animal sensorimotor function was highly variable, with subsets displaying a complete lack of response (top cluster), a partial response (middle cluster), or full restoration of function (bottom cluster). ND animals universally displayed a total lack of response to stimulation. (F) SKI gene expression, which is associated with peripheral myelination and glial cell proliferation, was up-regulated at 11 and 24 hpa but not at 7 dpa. ETV1 gene expression, which is associated with the peripheral glial repair response to injury, was down-regulated at 24 hpa and later up-regulated at 7 dpa. Means and SD are presented. ***P < 0.001.