Multimodal Single-Cell Analysis Reveals Disease-Specific Epithelial-Stromal Regulation of Inflammation in Periodontitis and Peri-implantitis

Abstract

Periodontitis and peri-implantitis are chronic inflammatory diseases in which dysregulated host-microbial homeostasis compromises the oral mucosal barrier and leads to progressive soft-tissue destruction and bone loss. Although epithelial and stromal compartments are positioned to regulate barrier integrity and immune infiltration, how these non-immune cells differentially mediate inflammation in periodontitis versus peri-implantitis remains incompletely understood. The present study tested the hypothesis that distinct inflammatory profiles in periodontitis and peri-implantitis are associated with condition-specific changes in epithelial and stromal cellular states and their interactions with immune cells. Soft-tissue biopsies were collected from adults with periodontal health (n = 5 sites), periodontitis (n = 7 sites), and peri-implantitis (n = 8 sites). Single-nucleus RNA sequencing generated a cell-resolved atlas across epithelial, stromal, and immune compartments. Differential abundance, gene set enrichment, and pseudotime analyses assessed condition-specific shifts in cell proportions, biological pathways, and epithelial differentiation trajectories. Spatial transcriptomics and multiplexed immunofluorescence were used to validate candidate epithelial and endothelial niches and to visualize proximity-based interactions in situ. An in vitro endothelial assay tested cytokine inducibility of MHC class II under pro-inflammatory stimulation. The epithelial compartment resolved into seven differentiation states spanning a pseudotemporal continuum from basal repair-associated cells to suprabasal barrier-forming cells. Periodontitis was characterized by expansion of least differentiated, repair-skewed epithelial states and loss of suprabasal barrier-forming states. Peri-implantitis retained suprabasal differentiation programs but showed enrichment of stress keratins (KRT6A/B/C and KRT16). CXCL14 expression localized predominantly to suprabasal epithelial cells and showed the strongest spatial association with immune proximity in periodontitis. In the stromal compartment, a rare HLA-DR/MHC II-positive endothelial subset was enriched in disease and localized adjacent to T cells. This phenotype was recapitulated in cultured human endothelial cells under pro-inflammatory stimulation. These results support a model in which epithelial differentiation state and endothelial immune adaptation contribute in distinct ways to inflammatory niche organization in periodontitis and peri-implantitis. By integrating high-resolution single-cell and spatial data, this work delineates cellular niches and regulatory signatures that distinguish these diseases and identifies barrier-associated and vascular programs relevant to future host-modulatory strategies.