1.Background

Aging is a gradual process in complex organisms in which various biochemical changes, cellular responses¹⁾, and gene activities affect in different aspects of tissue over time. Aging affects each individual differently, and even within an individual, each tissue can be affected diffe-rently. Therefore, studying aging at the organismal level is difficult. A typical problem is not only the genetic nonu-niformity of individual humans, but also the inability to distinguish between normal aging and the effects of various diseases throughout life²⁾. Hence, cellular senescence models are used in aging studies.

Cellular senescence is induced by various stimuli, inclu-ding passaging stress (telomere shortening), radiation, chemotherapy, oncogene activation, and oxidative stress, and it refers to stable cell cycle arrest. Aged cells accu-mulate deoxyribonucleic acid (DNA) damage owing to various stresses, reduce the function of various organelles within the cell, and alter various signal transduction systems and metabolic processes. These senescent cells secrete senescent cell-specific cytokines, chemokines, and growth factor, called senescence-associated secretion phe-notype (SASP)³⁾. This accelerates cell senescence or pro-motes the senescence of surrounding cells through auto-crine signaling or paracrine signaling.

Periodontal disease is classified as gingivitis or perio-dontitis, depending on the degree of severity. Gingivitis is a relatively mild and reversible form of periodontal disease and is limited only to the gingival tissue (soft tissue)⁴⁾. Periodontitis is a chronic inflammatory disease that is characterized by the destruction of periodontal tissue and loss of alveolar bone supporting the teeth. It is a common disease that occurs more than 90% of adults and is a major cause of tooth loss⁵⁾.

Various factors, including poor oral hygiene, systemic disease, drug use, and genetic factors, cause periodontal disease. The main risk factor for periodontal disease is periodontal pathogens (microorganisms of infection)⁶⁾. In a recent study, senescent cells were reported to induce inflammation in a number of chronic diseases (rheumatoid arthritis, atherosclerosis, and osteoporosis) through the induction of SASP and inhibit wound healing. However^3,7), the association between SASP and the mechanisms by which cell types in periodontal tissues undergo cellular senescence has not yet been fully elucidated.

2.Objectives

This study aimed to reveal the pathological role of cellular senescence in periodontal disease and investigate the possibility of regulating the expression of aging-and osteolysis-related factors in gingival fibroblasts. In this study, we investigated the effect of senescence induction in gingival fibroblasts on osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs).

1.Study design

Normal human gingival fibroblasts (HGFs) (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Corning, Glendale, AZ, USA) supplemented with 10% heat-ina-ctivated fetal bovine serum (Gibco Life Technologies, Grand Island, NY, USA) and 1% antibiotic–antimycotic solution. The HGFs were stimulated with 400 nM hydro-gen peroxide (Fujifilm Wako, Osaka, Japan) for 72 hours. The cells were incubated at 37°C in a humidified atmo-sphere containing 5% CO₂. The cells in which senescence was induced by hydrogen peroxide were assessed using a senescence-associated beta-galactosidase staining kit (SA- b-gal staining; Cell Signaling Technology, Danvers, MA, USA), in accordance with the manufacturer’s protocol. Positively stained blue cells were counted by counting three random areas under an ×100 magnification digital microscope (Olympus Corporation, Tokyo, Japan).

Protein expression was analyzed by western blotting. Harvested cells were lysed using Pro-prep (iNtRON Bio-technology, Seongnam, Korea) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein extracts were separated using sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Co., Milford, MA, USA). The membrane was blocked with 5% skim milk in phosphate-buffered saline containing 0.1%. Tween 20 for 1 hour at room temperature and probed with appropriate antibodies against p53, p16, p21, and beta- actin (Cell Signaling Technology). After washing, the membranes were incubated with horseradish peroxidase- conjugated anti-mouse or anti-rabbit immunoglobulin G secondary antibodies. Signals were developed using an enhanced chemiluminescence detection reagent (Bio-Rad, Hercules, CA, USA). IL-6, IL-8, IL-17, and osteocla-stogenic factors such as TNF-a and IL-1b concentrations in HGF cells culture supernatants were determined using enzyme-linked immunosorbent assays kits (R&D Sys-tems, Minneapolis, MN, USA) according to the manu-facturer’s protocol.

Mouse BMMs obtained from eight-week-old female mice (Charles River Laboratories, Seoul, Korea) were cultured in a-modified Eagle’s medium containing 10% fetal bovine serum for 24 hours. Then, non-adherent bone marrow cells were transferred to uncoated petri dishes with macrophage colony-stimulating factor (M-CSF, 30 ng/ml) for three days. To obtain the conditioned medium (CM) containing SASP factors. HGF cells were incubated with 400 nM hydrogen peroxide for 72 hours. Subse-quently, mouse BMMs were exposed to receptor activator of nuclear factor kappa-B ligand (receptor activator of nuclear factor kappa-B ligand [RANKL]; 50 ng/ml) or CM for five days. Mature osteoclast differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity. Osteoclasts were identified using TRAP kit (Sigma Aldrich, St Louis, MO, USA). Cells with three or more nuclei were scored as mature multi-nucleated osteoclasts. The absorbance was measured at 410 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

2.Data analysis and statistical methods

In this study, data are presented as the mean±standard deviation. To compare the expression of SASP and osteo-clast formation according to cellular senescence, a a-one- way analysis of variance was performed using SPSS 25.0 (SPSS Inc., Chicago, IL, USA), followed by Tukey’s post hoc analysis. Statistical significance was set at p-value of <0.05.

1.Upregulation of senescence-associated secretion phenotype in senescent human gingival fibroblasts

To investigate whether cellular senescence is related to the pathology of periodontal disease, the senescence cha-racteristics of gingival fibroblasts were examined. As oxidative stress is a major factor in aging, hydrogen peroxide was used as a stimulator to induce HGF cell senescence. The first, HGF cells were cultured for 72 hours after the addition of 400 nM hydrogen peroxide, and SA-b-gal staining was performed to confirm the degree of senescence. As shown in Fig. 1A, the proportion of SA-b-gal positive cells was observed to significantly increase (p<0.05).

Fig. 1. Characteristics of cellular senescence of human gingival fibroblasts (HGFs). HGFs were treated with 400 nM H₂O₂ for 72 h. (A) Senescence‐associated β‐galactosidase staining was performed to evaluate the degree of senescence in the HGFs. Scale bar=50 mm. (B) The cell cycle-related markers were measured using western blotting analysis. (C) Increased expression of senescent cell-specific cytokines and osteoclastogenic factors was determined by enzyme-linked immunosorbent assay. The values are expressed as the mean±standard deviation, and the experiments were repeated three times. *p<0.05 versus control.

Next, the expression of p53, p16, and p21, which are cell cycle-related markers of aging, was examined in HGF stimulated with 400 nM hydrogen peroxide. A significant increase was found in p53, p16, and p21 protein expre-ssion was observed in HGF, and the number of senescent cells was found to increase (Fig. 1B).

Subsequently, the expression levels of several SASP factors were examined to determine whether SASP, a hallmark of cellular senescence, was induced in hydrogen peroxide-treated HGF cells. The expression of IL-6, IL-8, IL-17, TNF-a, and IL-1b were found to be significantly higher in the experimental group than those in the control group (p<0.05) (Fig. 1C).

2.Hydrogen peroxide-induced human gingival fibroblasts senescence and osteoclastic differentiation

To examine whether, senescence indirectly regulates osteoclast differentiation, mouse BMMs treated with CM were cultured and prepared from hydrogen peroxide- treated HGF cells. A significant increase in osteoclast differentiation was found in cultured mouse BMM in CM compared to the control group (Fig. 2A). Moreover, oxidative stress-induced HGF cellular senescence increased the number of multinucleated TRAP-positive cells (Fig. 2B) and TRAP activity (Fig. 2C). Osteoclast formation was higher in the CM-treated experimental group than in the RANKL-treated experimental group (p<0.05).

Fig. 2. Indirect effects of cellular senescence of human gingival fibroblasts (HGFs) on osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs). HGFs were incubated with 400 nM H₂O₂ for 72 h, and the conditioned medium (CM) was collected. Mouse BMMs were cultured with receptor activator of nuclear factor kappa-Β ligand (RANKL) and CM as described in the Materials and Methods section. (A) After five days, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) as an osteoclast marker, and (B) the number of TRAP-positive multinucleated cells was scored. (C) TRAP osteoclast activity was assayed using TRAP cytochemical staining. Representative data from three experiments are shown. Scale bar=100 mm. Values are expressed as mean±standard deviation. The control group was comprised of the macrophage colony-stimulating factor treatment group. *p<0.05 versus the RANKL treatment group.

1.Interpretation

The host inflammatory response can cause periodontal tissue destruction⁴⁾. Bacteria alone are not sufficient to promote the development of periodontal disease, but bacterial infection is necessary to facilitate the initial inflammatory response⁶⁾. Continuous infiltration of immune cells is induced by continuously produced pro-inflammatory mediators, which can eventually lead to collateral damage to healthy tissue⁸⁾. Previous studies reportied that the destruction of periodontal tissue, including resorption of alveolar bone, occurred in both old germ-free mice (12 to 30 months) and rats (18 months)^9,10). These results suggest that age-associated factors may contribute to the severity of periodontal destruction as aging progresses. This study investigated the expression of aging-and osteolytic-related molecules in gingival fibroblasts.

At the cellular level, aging is a phenomenon that occurs when abnormal cells continue to perform abnormal acti-vities without being easily removed, and the root cause of each cell’s abnormality is ‘cell damage’. DNA damage can induce aging phenotypes, including stimuli of ionizing radiation, oxidative stress, and lipopolysaccharid¹¹⁾. Aging at the individual level accompanies physical loss, functional deterioration, and various aging-related diseases. Under-standing aging at the cellular level is essential for under-standing aging at the individual level, and recent research has highlighted the importance of cellular senescence.

Periodontal disease is a chronic inflammatory disease that is comorbid with other age-related diseases; however, the underlying mechanisms have not yet been elucidated. In particular, the occurrence and severity of periodontal destruction have been shown to increase with age¹²⁾. Oxidative stress has been reported to induce gingival fibroblast senescence and the effect of alveolar bone cell senescence on periodontal disease¹³⁾. Kiyoshima et al.¹⁴⁾ reported higher levels of SA-b-gal, p16, p21, and pro‐inflammatory cytokine expression in HGF cells incubated for five days after hydrogen peroxide stimulation. Senescent cells induced the secretion of various inflammatory cyto-kines, chemokines and matrix metalloproteinases, suggesting that these factors induce chronic inflammation^11,15,16).

In this study, we confirmed that oxidative stress increa-sed the expression of the cellular senescence markers, IL-6, IL-8, and IL-17 in senescent HGF cells. Notably, previous studies have found that SASP proteins promote cell senescence by acting on the original or neighboring cells through autocrine or paracrine mechanisms^17,18). Kuilman et al.¹⁹⁾ also found that SASP inhibits the proli-feration of surrounding normal cells and even promotes cancer progression. In our experiments, senescent HGF cells produced a representative SASP protein (Fig. 1). SASP factors in senescent HGF cells, including inflam-matory cytokines and chemokines, may have major effects on inflammation and the destruction of senescent perio-dontal tissue. Moreover, our results showed that osteoclast differentiation was promoted by the hydrogen peroxide- induced senescent of HGF (Fig. 2). Indirectly, senescent cells can also induce the upregulation of pro-inflammatory cytokines found in periodontitis, consistent with the findings of Chen et al.²⁰⁾ that osteoclastogenesis is induced through the RANKL-Receptor activator of nuclear factor kB, Osteoprotegerin pathway. These results suggest that the secreted factors of senescent cells interact with cells in an autocrine or paracrine manner.

2.Limitations

This study has several limitations. It was designed and performed in vitro using gingival fibroblast cells, a type of periodontal tissue. In this study, it was unclear whether and how the SASP in the senescence of oxidative stress- induced HGF cells played a negative role in vivo by accelerating the senescence of surrounding cells due to SASP secreted from senescent cells. In addition, an indirect approach was used to identify the affect of senescent cells on inflammatory osteolytic activity. This is an important issue that should be examined in future studies. Follow-up studies are required to examine the mechanism by which the detrimental function of senescent cells is suppressed during the pathogenesis of periodontal disease.

3.Generalizability

This study demonstrates that the expression of senescence-related factors is upregulated in gingival cells stimulated by oxidative stress and contributes to osteocla-stogenesis in vitro. Therefore, understanding the patholo-gical role and function of cellular senescence in perio-dontal disease can help to provide insights into periodontal tissue inflammation and destruction.

4.Suggestions

In summary, we investigated the association between senescence-related factors and osteoclast differentiation in gingival cells after senescence induction. The results demonstrated that the production of SASP associated with senescent gingival cells increased, and more importantly, the activity of osteoclasts, including bone resorption, increased, indicating the possibility of more severe perio-dontal tissue destruction. Therefore, the development of potential therapies to treat periodontal and age-related diseases by targeting specific SASP factors could be an effective treatment strategy for the elderly population.

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2022R1F1A1076563).

No potential conflict of interest relevant to this article was reported.

This project does not require IRB review because it is an experimental paper using commercially available cells.

Please contact the corresponding author for data avai-lability.