Molecular Signatures of Senescence in Periodontitis: Clinical Insights

Abstract

Most of the elderly population is afflicted by periodontal diseases, creating a health burden worldwide. Cellular senescence is one of the hallmarks of aging and associated with several chronic comorbidities. Senescent cells produce a variety of deleterious secretions, collectively termed the senescence-associated secretory phenotype (SASP). This disrupts neighboring cells, leading to further senescence propagation and inciting chronic inflammation, known as "inflammaging." Detrimental repercussions within the tissue microenvironment can trigger senescence at a younger age, accelerate biological aging, and drive the initiation or progression of diseases. Here, we investigated the biological signatures of senescence in healthy and diseased gingival tissues by assessing the levels of key senescence markers (p16, lipofuscin, and β-galactosidase) and inflammatory mediators (interleukin [IL]-1β, IL-6, IL-8, matrix metalloproteinase [MMP]-1, MMP-3, and tumor necrosis factor-α). Our results showed significantly increased senescence features including p16, lipofuscin, and β-galactosidase in both epithelial and connective tissues of periodontitis patients compared with healthy sites in all age groups, indicating that an inflammatory microenvironment can trigger senescence-like alterations in younger diseased gingival tissues as well. Subsequent analyses using double staining with specific cell markers noted the enrichment of β-galactosidase in fibroblasts and macrophages. Concurrently, inflammatory mediators consistent with SASP were increased in the gingival biopsies obtained from periodontitis lesions. Together, our findings provide the first clinical report revealing susceptibility to elevated senescence and inflammatory milieu consistent with senescence secretome in gingival tissues, thus introducing senescence as one of the drivers of pathological events in the oral mucosa and a novel strategy for targeted interventions.

Keywords: aging; cellular senescence; inflammaging; inflammation; periodontal diseases; senescence-associated secretory phenotype.

Figure 1.

P16 levels in the gingival tissues of healthy and periodontitis subjects. (A) Comparison of relative mRNA expression of P16 between healthy sites (n = 31) and periodontitis lesions (n = 44). (B) Relative mRNA expression of P16 in healthy and periodontitis samples from both young and aged populations (healthy young: n = 21, healthy aged: n = 10, periodontitis young: n = 20, periodontitis aged: n = 24). (C) Representative immunofluorescence images of p16 (red) in human gingival tissues from healthy and periodontitis aged subjects (magnification 40×). (D) Quantification of the relative fluorescence intensity of p16 (healthy: n = 7, periodontitis: n = 9). Individual data points are plotted, and the averages are shown. The difference between the 2 groups was statistically analyzed by Mann-Whitney U test. Kruskal-Wallis with post hoc Dunn’s test was performed for multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 2.

Lipofuscin deposition in the gingival tissues of healthy and periodontitis subjects. (A) Quantification of Sudan Black B (SBB)–positive cells/field between healthy sites (n = 15) versus periodontitis lesions (n = 22). (B) Representative images of gingival tissue biopsies from healthy young, periodontitis young, healthy aged, and periodontitis aged patients stained with SBB (magnification 40×). White arrows indicate SBB-positive cells. (C) Quantification of SBB-positive cells/field stratified by age (healthy young: n = 8, healthy aged: n = 7, periodontitis young: n = 10, periodontitis aged: n = 12). Individual data points are plotted, and the averages are shown. The difference between the 2 groups was statistically analyzed by Mann-Whitney U test. Kruskal-Wallis with post hoc Dunn’s test was performed for multiple comparisons. *P ≤ 0.05, ***P ≤ 0.001. Ge, gingival epithelium; Ct, connective tissue.

Figure 3.

SA-β-galactosidase levels in the gingival tissues of healthy and periodontitis subjects. (A) Quantification of the percentage of the SA-β-galactosidase–positive area between healthy sites (n = 10) and periodontitis lesions (n = 10). (B) Representative images of gingival tissue biopsies from healthy young, periodontitis young, healthy aged, and periodontitis aged patients stained with SA-β-galactosidase (magnification 20×; healthy young: n = 5, healthy aged: n = 5, periodontitis young: n = 5, periodontitis aged: n = 5). (C) Quantification of the percentage of SA-β-galactosidase–positive area stratified by age (magnification 20×). (D) Representative immunofluorescence images of β-galactosidase (green) and CD68 (red) in human gingival tissues from healthy and periodontitis subjects (magnification 40×). (E) Quantification of the percentage of colocalization of β-galactosidase and CD68 (healthy: n = 8, periodontitis: n = 11). (F) Representative immunofluorescence images of β-galactosidase (green) and TE7 (red) in human gingival tissues from healthy and periodontitis subjects (magnification 40×). (G) Quantification of the percentage of colocalization of β-galactosidase and TE7 (healthy: n = 8, periodontitis: n = 13). Individual data points are plotted, and the averages are shown. The difference between the 2 groups was statistically analyzed by unpaired t test (A, E) and Mann-Whitney U test (G). One-way analysis of variance with Tukey’s post hoc test was performed for multiple comparisons (C). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 4.

Inflammatory mediators consistent with SASP in the gingival tissues of healthy and periodontitis subjects. Relative mRNA expression of IL-1β (healthy young: n = 8, healthy aged: n = 7, periodontitis young: n = 19, periodontitis aged: n = 21) (A, B), IL-6 (healthy young: n = 8, healthy aged: n = 7, periodontitis young: n = 15, periodontitis aged: n = 21) (C, D), IL-8 (healthy young: n = 14, healthy aged: n = 6, periodontitis young: n = 19, periodontitis aged: n = 21) (E, F), TNF-α (healthy young: n = 8, healthy aged: n = 7, periodontitis young: n = 18, periodontitis aged: n = 21) (G, H), MMP-1 (healthy young: n = 8, healthy aged: n = 7, periodontitis young: n = 19, periodontitis aged: n = 21) (I, J), and MMP-3 (healthy young: n = 8, healthy aged: n = 7, periodontitis young: n = 19, periodontitis aged: n = 21) (K, L) in healthy and periodontitis samples from both young and aged. Individual data points are plotted, and the averages are shown. The difference between the 2 groups was statistically analyzed by Mann-Whitney U test. Kruskal-Wallis with post hoc Dunn’s test was performed for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5.

Conceptual model illustrating the impact of cellular senescence on periodontal health. The interplay between chronic inflammation, senescence, and host susceptibility can collectively establish a damaging cycle, disrupting tissue homeostasis. Elevated p16 expression signals a tendency toward cell cycle arrest, hindering cellular function and regenerative capabilities. Increased levels of β-galactosidase and lipofuscin deposition, coupled with heightened inflammation, imply metabolic and lysosomal alterations. As a result, cellular senescence likely plays a substantial role in the pathophysiology of periodontal diseases.