Fig 2: Fibroblasts activated to guide leukocyte migration (AG fibroblasts) are one of three fibroblast subpopulations in gingival tissue during periodontitis development. (A) Violin plots showing gene expression levels of type I collagen (Col1a1), type XIV collagen (Col14a1), and smooth muscle aortic actin 2 (Acta2) in gingival fibroblast subpopulations during periodontitis development. AG, AG fibroblasts; KT, ‘keeping typical phenotype’ fibroblasts; MF, myofibroblasts. Gene ontology (GO) enrichment analysis of the biological functions of AG fibroblasts and KT fibroblasts on Day 0 without ligature placement (B) and on Day 1 (C) and Day 7 (D) after ligature placement. Gene clusters related to immune regulation (red) were identified in AG fibroblasts, and these clusters dominate after ligature placement. (E) t-SNE projection plots showing fibroblast subpopulations in gingival tissue during periodontitis development. Colors indicate cell type, as follows: blue, KT fibroblasts; red, AG fibroblasts; and yellow, MFs. (F) Proportion plots showing the relative amounts of each fibroblast subpopulation on Days 0, 1, 4, and 7.

Fig 3: AG fibroblasts and immune surveillance in periodontitis development. (A) Dot plots depicting expression levels of the CCL genes Ccl8, Ccl11, Ccl19, Cxcl1, Cxcl9, Cxcl10, and Cxcl12 in gingival fibroblast subpopulations during periodontitis development. (B) Dot plots depicting expression levels of the Toll-like receptor and related genes Tlr2, Tlr3, Tlr4, Myd88, Irak1, Map3k7, and Rela in gingival fibroblast subpopulations during periodontitis development. Upregulation of chemokines and TLR-related molecules is predominantly observed in the AG fibroblast subpopulation. (C) Hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining for COL14A1 and CXCL12 in periodontal tissue on Day 1; scale bars, 100 µm (HE) and 20 µm, (IHC). Yellow arrows indicate COL14A1- and CXCL12-positive cells in the connective tissue papillae and periodontal ligament (PDL).

Fig 4: Bar graph representation of identified various collagen types across study groups. (A–E) Fibril-forming collagens: includes types such as -1, -2, -3, -5, and -11. Significant higher expression of COL1A1 was observed (fetal disk phenotype [FD] vs. normal disk phenotype [ND], p=0.0281; FD vs. degenerated disk phenotype [DD], p=0.0005) and COL1A2 in FD group (FD vs. DD, p=0.0019). COL2A1 was highly expressed in FD group with p-value, 0.0123 and with ND group (p=0.0043). Also, COL11A2 was found with increased expression in FD group against ND (p<0.0001) and DD (p=0.0002) groups, respectively. (F–K) FACIT collagen: includes types such as -9, -12, -14, -16, -19, and -20. Expression of COL9A1, COL12A1, and COL14A1 was found higher in FD group. Specific expression of COL16A1 and COL19A1 was found in herniated disk phenotype (DH) and scoliotic disk phenotype (SD) group. COL20A1 was found higher in SD followed by DH group. (L–O) Other types of collagens: includes microfibrillar/beaded filament collagen, network-forming collagen and multiplexins. COL6A1 was found higher in SD group; whereas COL6A2 and COL6A3 higher in FD comparatively. COL6A3 showed significantly higher expression against ND (p=0.0002). COL10A1 was higher in DD followed by DH. Multiplexin-COL15A1 was specific to FD whereas COL18A1 was higher in DD followed by DH. Y-axis represents average normalized spectral counts and the x-axis represents study group. (*) indicates statistical significance corrected using multiple statistical tests in IBM SPSS ver. 25.0 (IBM Corp., Armonk, NY, USA) with color codes (FD vs. ND, red; ND vs. DD, green, and FD vs. DD, violet).

Fig 5: Validation of COL14A1 by immunohistochemistry. Ten fetal samples and five normal disk phenotype samples were used. Seven of fetal samples showed strong cytoplasmic and extracellular matrix (ECM) staining. In contrast, only two adult samples showed mild staining in ECM (magnification ×400, scale bar=50 µm). Positive samples are stained brown.