Multi-omics characterization of fibroblast subpopulations involved in osteogenic differentiation in periodontitis

Background Fibroblasts are the predominant stromal cells in periodontal tissues and are involved in inflammation and repair. However, their heterogeneity and molecular mechanisms remain poorly defined. Our study is to identify fibroblast subpopulations driving osteogenesis in periodontitis and their molecular markers. Methods We integrated bulk RNA sequencing with public datasets to select osteogenesis-related genes in periodontal ligament fibroblasts. Single-cell RNA sequencing resolved fibroblast heterogeneity; pseudotime trajectory inference (Slingshot and Monocle 2) and tradeSeq explored differentiation dynamics and hub genes. In silico knockout analysis predicted effects of marker loss-of-function. Functional enrichment explored cell subpopulation functions and perturbation gene effects. CellChat analyzed intercellular crosstalk. Spatial transcriptomics mapped subpopulations’ spatial enrichment within the tissue. Marker expression was validated by RT-qPCR and immunostaining in cultured cells and mouse models. Results 14 core osteogenesis-related genes were identified, with CCN4 and DPT being specifically identified as markers of fibroblast subsets, along with three other populations. Pseudotime analysis revealed divergent differentiation trajectories of CCN4 + fibroblasts in health v.s. periodontitis: in health they gave rise to immune-functional Fibroblasts 2, whereas in periodontitis they yielded osteogenic Fibroblasts 3. During osteogenic differentiation in periodontitis, 19 and 94 genes were identified in CCN4 + and DPT + fibroblasts, respectively. In silico knockout of CCN4 and DPT perturbed genes enriched in osteogenesis and angiogenesis pathways. Intercellular crosstalk among CCN4 + , DPT + fibroblasts, and endothelial cells involved WNT, MK, NOTCH and PDGF, with MK dominated fibroblast-fibroblast communication. Spatial proximity was highest among CCN4 + , Fibroblasts 1, DPT + and Fibroblasts 3, as well as between DPT + and endothelial cells, while Fibroblasts 2 were distant. In vitro and in vivo experiments confirmed that CCN4 and DPT were highly expressed under osteogenic and periodontitis conditions. Conclusion CCN4 and DPT are signature genes of fibroblast subpopulations with osteogenic differentiation in periodontitis, potentially contributing to tissue repair through key intracellular and intercellular molecules.