166. Understanding The Mechanisms Driving Hypertrophic Skin Scarring In Mice Using Single Cell Rna Sequencing

INTRODUCTION: Hypertrophic scarring (HTS) is a dermal form of a fibroproliferative disorder that leads to considerable morbidity. No effective treatment currently exists to overcome HTS due to limited understanding cellular mechanisms underlining HTS formation. In this study, we utilized a mouse model of HTS to investigate the fibroblast populations and molecular pathways responsible for HTS. METHODS: We applied HTS skin devices to adult black six mice (6-8 weeks of age) for 14 days. At post-operative 14 (POD 14) we processed the tissue into single cell suspension for fluoresce-activated cell sorting (FACS). Using a lineage negative gating FACS strategy, we isolated the fibroblasts from the cell suspension and then submitted the cells for 10X single cell RNA sequencing, using the 10X platform. Fibroblasts from unwounded skin was also processed for sequencing as a control. We further compared our mouse model histologically to human hypertrophic scars using H&E and picrosirius red staining to evaluate how closely our model resembles human HTS. RESULTS: Histologically, at POD 14, H&E staining of the mouse HTS showed a similar architecture to human HTS. In addition, analysis of extracellular matrix collagen ultrastructure with picrosirius red staining showed a distinct mouse HTS profile similar to human HTS. Using the Seurat analytical pipeline, we identified distinct fibroblast subpopulations in the mouse HTS scars compared to the unwounded skin. Gene ontology pathways using the Enrichr tool, showed a predominance of mechanical signaling pathways in the fibroblasts derived from HTS compared to unwounded fibroblasts. The mechanosensitive fibroblasts in the mouse HTS scars expressed distinct cell surface markers compared to unwounded skin. CONCLUSION: We have identified mechano-sensitive fibroblast subpopulations responsible for HTS in mice. Future studies evaluating the targeted modulation of these mechanical active fibroblast subpopulations, may provide targets to overcome human HTS.