Cellular heterogeneity in hypertrophic burn scars in response to carbon dioxide laser therapy

Background: Ablative fractional carbon dioxide (AFCO2) laser therapy is used for treating pathological scarring. However, mechanisms underlying reduction in hypertrophic scarring are poorly understood. Methods: We investigated cellular mechanisms of AFCO2 laser therapy by performing single-cell RNA sequencing (scRNA-seq) on skin biopsies from burn survivors with hypertrophic scars before and after three sessions of AFCO2 therapy. Scar reduction was assessed subjectively and objectively. Results: Those with a good response (GR) to laser therapy have scars less than 6 years from injury, whereas poor responders (PR) have scars over 6 years since injury. ScRNA-seq analysis of skin biopsies reveals that genes enriched in GR are associated with extracellular matrix and structure organisation (COL14A1, POSTN, SPARC); whereas genes enriched in PR are related to enhanced immune inflammatory responses (CXCL14, JUN, TNC). Notably, expression of the pro-fibrotic gene Engrailed-1 (EN1) remains elevated in PR scars compared to GR. The ECM-regulatory gene TIMP-1 (Tissue Inhibitor of Metalloproteinases 1) is also significantly upregulated in PR scars following treatment relative to GR scars, although at the protein level TIMP1 levels decrease in PR but increase in GR after therapy. The regenerative-associated gene, TRPS1 (Transcriptional Repressor GATA Binding 1) expression demonstrates opposing regulation post treatment, with upregulation observed in GR scars but downregulation in PR scars, underscoring distinct transcriptional trajectories associated with differential therapeutic outcomes. Finally, distinct intercellular communication networks and differentiation trajectories are observed after AFCO2, with regenerative mesenchymal fibroblasts predominating in GR but inflammatory fibroblasts associated with PR. Conclusions: We conclude AFCO2 laser therapy is more effective if done early after injury and distinct fibroblast recruitment is associated with a good response, specifically regenerative fibroblasts. Plain English summary: Ablative fractional carbon dioxide (AFCO2) laser therapy is used for reducing scarring. However, results are varied and the factors associated with a good response are poorly understood. We assessed scar reduction after AFCO2 therapy in patients with excessive scarring, taking skin biopsies to determine the cell types present in the treated areas and which genes they expressed. We showed that the therapy was more effective in patients who were less than 6 years from injury, those with a good response had scars containing regenerative fibroblasts and gene expression that was distinct from those with a poor response. We conclude that AFCO2 therapy is most effective if used early after injury and that a more regenerative, less inflammatory cell infiltration is associated with scar reduction.