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The Oral Mucosa Hosts Distinct Fibroblast Subpopulations To Facilitate Regenerative Wound Repair
Michelle Griffin, MD PHD1,2, Jessica Cook, BS3, Dario Bofelli, PHD3, Michael Januszyk, MD PHD1, Ophir Klein, MD PHD3, Michael Longaker, MD1.
1Stanford University, Stanford, CA, USA, 2University College London, London, United Kingdom, 3University of California, San Francisco, CA, USA.

PURPOSE:The oral mucosa is a unique tissue that displays regenerative scarless healing following wounding in under 3 days. Growing evidence has aimed to characterize the fibroblasts responsible for scarring following skin injury in the hope to modulate skin scarring and fibrosis. However, the identification and behavior of oral mucosal fibroblasts (OMFs) that facilitate regeneration are unknown. This observation presented a unique opportunity to study the molecular events differentiating oral regeneration from skin scarring.
METHODS:To identify the OMF subpopulations responsive to dermal wounding, we examined the transcriptional responses of OMFs to mechanical injury in a novel mouse model. Adult C57Bl/6 mice received matched 2-mm wounds within their buccal mucosa and facial skin to allow for simultaneous evaluation of fibroblasts subgroups. At 1-, 2-, 4- or 7-days post-wounding, mice were euthanized and whole tissues was analyzed for histology, fluorescence-activated cell sorting (FACS) and confocal microscopy (n=10). At the similar timepoints wounds were prepared for single-cell RNA sequencing, including unwounded samples (n=10). Using R and Python software packages, we defined the fibroblast subtypes in oral wounds in comparison to skin wounds at all timepoints. Furthermore, we identified and defined the cross-talk between cell types in oral and skin wounds using network analysis platforms.
RESULTS:Our dataset identified dynamic changes in the cell composition of the wound environment in the oral and skin tissue including the epithelial, mesenchymal and immune populations following wounding. Our analysis revealed that the fibroblast cells clustered differently in the oral and skin tissue following injury. Cluster analysis reveals five subpopulations of fibroblasts with unique cluster markers in the oral tissue following wounding. Pseudotime analysis revealed that the fibroblasts following skin and oral injury followed distinctly unique transcriptional trajectories. Wnt1+ positive oral fibroblasts were identified in a subset of the oral mucosa to a greater degree than in the facial skin. To evaluate these fibroblasts a lineage tracing mouse model where Wnt1+ cells are marked as GFP+ was developed. Histology and FACS of Wnt1CreER;ROSA26mTmG mice wounds (Fig.1A) at 4 days post-wounding confirmed that infiltration of Wnt1+ cells in the oral mucosa compared to the skin and unwounded tissue (P < 0.05) (Fig.1B-C). Positive staining of collagen type I and FACS staining confirmed the infiltrating Wnt1+ cells in the oral mucosa to be fibroblasts (Fig.1B-C). Cell-cell interactions were also notably different between the oral and skin tissue with specific upregulation and downregulation of pathways as a result of wounding in the oral tissue.
CONCLUSION:These data suggest that oral tissue displays regeneration following wounding due to its unique resident population of fibroblasts. Studies to examine and mimic the behavior of these Wnt1+ oral regenerative fibroblast subpopulations hold promise to unlocking the mechanism of scarless wound repair.


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