Purpose: Dermal wound healing is comprised of multiple phases: coagulation, inflammation, cell proliferation, and remodeling. During remodeling, the temporary matrix deposited during the proliferation phase is gradually replaced by one that is more structured and organized, and is considered critical for proper wound healing. Dysregulation in remodeling can occur due to many variables including ischemia, infection, and radiation, and may lead to pathologic healing. Previous work from our group and others have investigated the early stages of wound healing in depth; however, molecular investigations into the cell types and signaling mechanisms involved in the later-stage remodeling phase have not yet been conducted. This gap presented a unique opportunity to study the late stage of wound healing using a multi-omic approach.
Methods: Adult C57Bl/6 mice received 8-mm stented dorsal excisional wounds, which were harvested at days 2, 7 and 14 (early-stage wound healing), and days 60, 105 and 150 (late-stage wound healing). Unwounded skin served as controls. Whole tissues were analyzed for histology, confocal microscopy, and CODEX multiplexed imaging. Wounds from the same timepoints were also prepared for Chromium Single Cell RNA sequencing (scRNAseq), and spatial transcriptomics. Using software packages from R and Python, we identified and defined fibroblast subtypes in wounds at early and late-stage wound healing. Combining CODEX, single-cell and spatial transcriptomics, crosstalk and signaling pathways were further identified via network analysis platforms.
Results: Grossly, after wound closure at approximately 14 days, scars from these initial wounds remained visible 150 days following wounding. On histologic anlysis, remodelled wounds continued to exhibit characteristic features of early-stage scars, including an absence of hair follicles and other skin appendages. Interestingly, ultrastructure analysis of Picrosirius red staining illustrated distinct separation of early and late-stage scars, with both groups spatially separating from unwounded skin. Notably, similar to what has previously been oberserved in the earlier stages of wound healing wherein mechanotransduction pathways play a critical role, spatial transcriptomic and proteomic analyses confirmed the continued importance of mechanical signaling pathways remained upregulated in remodelled wounds. These data together suggest that dynamic cellular and molecular changes occur during the remodeling phase of wound healing. Spatial proteomic and transcriptomic analysis confirmed that cells within the wound environment differed in their composition and distribution between early and late-stage wound healing.
Conclusion: These results demonstrate that the remodeling phase of wound healing remains a dynamic process with cellular and signaling compoents within the tissue driving continued maintenance of dermal fibrosis even months following injury. Further investigations into these cell types and signaling pathways hold promise in identifying therapies for wounds where remodeling is dysregulated, and improve wound healing in clinical settings.