Repair of the Murine Tympanic Membrane Displays Hallmarks of Regeneration

Abstract

BackgroundThe tympanic membrane (TM) has a remarkable ability to repair itself, with perforations typically closing in days to weeks in all mammalian species studied. The cellular and molecular mechanisms underlying TM repair remain largely unknown, however. This study looked to thoroughly characterize the repair process of the injured TM and uncover mechanisms of repair to inform future treatments for TM disorders. We hypothesized that the TM does not undergo canonical “wound healing” but regenerates in response to injury.MethodsCells from TMs injured on adult mice 14, 7, 3, and 1 day prior to sacrifice were submitted for single‐cell RNA sequencing using the 10x Genomics scRNA‐seq platform. Using Seurat clustering and the CellFindR algorithm, unbiased cell clusters were generated with matrices of differentially expressed genes for each cluster. Protein detection via immunofluorescence (IF) and RNA detection via RNAscope were used to biologically validate and define the anatomic locations of distinct cellular populations. The K5Cre‐ERT2 promoter was utilized in mouse models to genetically manipulate and fluorescently label keratinocyte (KC) populations. The mTmG and Confetti reporters were utilized as fluorescent labels.ResultsRNA expression data from all timepoints of perforation were merged and analyzed, revealing 8 distinct major populations of cells and revealing time‐dependent transcriptional shifts in each layer of the TM. From both cross‐sectional and whole‐mount views, the TM shows a rapid, proliferative response to injury by 18 hours post‐injury, predominantly in the KCs. 3 days after perforation, there are large transcriptional shifts in the immune, mesenchymal, and mucosal populations. The multi‐layered tissue shows a large volumetric increase by day 7 but quickly remodels and restores the original volume of the TM by day 14. At slightly longer timepoints, the radial and circular collagen patterning of the TM is also restored, creating a scar‐free structure. We identified a regeneration‐induced “wounded epithelial” population, characterized by a combination of distinct marker genes. A K5Cre‐ERT2;Confetti mouse model shows that the population migrates from known stem cell regions of the organ to the site of injury. Based on expression values and immunostaining, EGFR signaling is upregulated during regeneration, corresponding with increased expression of EGFR ligands and processing co‐factors. When EGFR is deleted in vivo, using a K5‐CreERT2;Egfrfl/fl; R26mTmG/mTmG mouse model, TMs no longer display proliferation post‐injury and cannot repair perforations.ConclusionsTM perforation healing displays a complex and coordinated series of transcriptional, morphological, and patterning events, ultimately culminating in the restoration of normal tissue architecture to the TM. These characteristics are more typical of epimorphic regeneration, rather than more typical scar induced mammalian wound healing.