M. Griffin1, J. Cook2, D. Boffelli2, R. Zwick2, D. Wan1, O. Klein2, M. Longaker1 1Stanford University, Plastic And Reconstructive Surgery, Palo Alto, CA, USA 2University Of California – San Francisco, Department Of Pediatrics, San Francisco, CA, USA
Background
The oral mucosa (OM) repairs wounds more efficiently than any other barrier site in the adult human body, allowing for scarless healing in just 1-3 days. Rapid re-epithelialization is a hallmark of the tissue, yet the cellular and molecular mechanisms of regeneration in the OM remain unclear. We sought to identify the heterogenous population of fibroblasts in the OM in comparison to those in the skin, probing both the identities and functions of the oral mucosal fibroblast (OMF) population and the mechanisms they utilize to facilitate scarless wound healing over fibrosis.
Methods
To define the identities of OMF subpopulations, we examined the transcriptional responses of murine OMFs and dermal fibroblasts (DFs) to mechanical injury using single-cell RNA sequencing, Visium spatial RNA analysis and CODEX spatial protein analysis. Unwounded tissue was collected along with tissue 1-, 2-, 4- or 7-days post-wounding from both tissues and used to define OMF subtypes and their signaling profiles.
Results
Histology by H&E demonstrated that by post-operative day (POD)-4 oral mucosal wounds had repaired, such that the tissue architecture resembled that of unwounded oral tissue. In contrast, at POD4 facial skin wounds appeared distinct from the control, and they healed with a scar by POD7 (Fig. A). Our scRNA dataset showed distinct cell populations from multiple compartments of the tissue, including populations from the epithelium, mesenchyme, and immune system (Fig. B). Using packages in both R and Python, we clustered the cells and performed network analysis to establish exact pathways and gene networks that are up-regulated due to wounding in each tissue. Our analysis reveals nine novel oral mucosal fibroblast subpopulations with distinct cluster markers and putative functions, together with several pathways related to wound response utilized uniquely by the OM in comparison to the skin (Fig. C). In vivo pharmacological perturbation and genetic tracing of one pathway of interest reveals this signaling network as necessary for rapid wound healing in the OM. Immunohistochemistry of human oral and skin scars (n=6) confirmed similar signaling networks as those identified in murine wounds.
Conclusions
Collectively, this work interrogates the process of scarless regeneration in the OM at a granularity never previously seen within the field and reveals the divergent molecular programs that differentiate skin and oral wound healing.
