Hyaluronic acid networks and emergent tissue mechanics orchestrate mammalian limb regeneration

In mammals, only distal amputations of the terminal digit regenerate, while more proximal ones result in non-regenerative, fibrotic scarring, underscoring the limits of our regenerative capacity. The mechanisms that direct one wound healing process over the other remain poorly understood. In this study, we investigated the role of cell-extrinsic cues, focusing upon the role of the extracellular matrix (ECM) and the bulk mechanical properties that arise from its composition. Through single-cell RNA sequencing (scRNA-seq), we showed that non-regeneration and regeneration are differentially dominated by a select few mesenchymal cell sub-types that help determine the mechanical properties of their environment by synthesizing collagen remodeling proteins and glycosaminoglycans. In the regenerating blastema, we demonstrated that osteo-lineage cells deposit copious networks of hyaluronic acid (HA) and closely associated proteins, which corresponded with soft tissue mechanical properties. Using synthetic stiffness-tunable scaffolds, we further demonstrated that fibroblast-ECM feedback mechanisms exist, in which soft mechanical cues mimicking the blastema propagate the synthesis pro-regenerative ECM. Knockdown of HA networks critically impairs regeneration, promotes fibrotic viscoelastic mechanical properties, and enhances collagen fibrillogenesis. We further showed that stiffness affects regeneration by modulating the strength of early BMP signaling. Lastly, we stabilized the deposition of HA networks in vivo using Hyaluronic Acid and Proteoglycan Link Protein 1 (HAPLN1) to partially rescue non-regenerative amputations. Altogether, we showed that through feedback mechanisms, the ECM and emergent bulk tissue mechanics mediate mesenchymal cell activity during injury repair. These findings demonstrate that modulating the ECM microenvironment may augment restorative repair and inform future therapeutic strategies to overcome deficiencies in mammalian wound healing. Overall design: Non-regenerative amputations were performed on adult wild type C57BL/6 mice at the second phalanx, and the single cell transcriptome was compared with datasets of regenerative amputations. To investigate the influence of HA networks in regeneration, mice were fed either control diet or 4-methylumbelliferone-infused diet (50g/kg), which globally depletes the digit of HA and Aggrecan. Regenerative amputations were subsequently conducted, and cells were harvested from the digits at 14DPA for scRNAseq (N=2 per condition). All datasets were generated using the 10X Genomics platform. Raw and processed data are provided.