Analysis of cellular senescence in gingival tissues and gingival fibroblast cultures


 Senescence in periodontal tissues may be involved in the pathogenesis of periodontitis. In this study, we analyzed cellular senescence in gingival tissues of aged mice and in gingival fibroblast cultures and investigated the relationship of cellular senescence with the pathogenesis of periodontal disease. The proportions of senescence-associated β-galactosidase (SA-β-gal)-positive cells and the mRNA expression levels of p16 and p21 in gingival tissues and gingival fibroblasts from 20-month-old mice were significantly higher than those in gingival tissues and gingival fibroblasts from 10-week-old mice. The mRNA expression levels of IL-1β and TNF-α as well as the numbers of total macrophages and M1 macrophages were increased in gingival tissues of aged mice. Expression of these senescence-associated molecules was also increased in long-term passaged or hydrogen peroxide-stimulated human gingival fibroblasts; however, it was significantly suppressed by treatment with the senolytic drug ABT-263. These results suggest that cellular senescence can be induced in gingival fibroblasts in culture as was observed in gingival tissues of aged mice. An increase in senescent cells in the gingiva may be associated with the pathogenesis of periodontitis in part through activating an inflammatory response in periodontal tissues.


Introduction
Aging is the most powerful risk factor that is common to chronic diseases and increased mortality. 1 It has been suggested that aging also contributes to the development of lifestyle-related diseases such as cardiovascular diseases and diabetes, and aging of cells in blood vessels and adipose tissue may be the basis of the pathogenesis of lifestyle-related diseases. [2][3][4][5] Periodontitis is also a disease for which the incidence increases with aging, and aging is thought to be one of the risk factors of periodontitis. 2 However, the relationship between aging of periodontal tissues and the pathogenesis of periodontitis has remained unclear. 3 Senescence at the cellular level (cellular senescence) 4 in organ tissues not only affects the senescence of individuals but may also be involved in the development of various age-related diseases. Cellular senescence is a phenomenon 5 in which irreversible cell cycle arrest occurs, and it is caused by telomere shortening, regulation of oncogenes and tumor suppressor genes, and stresses due to radiation, ultraviolet rays, and reactive oxygen species. Senescent cells share common characteristics including cell cycle constancy, cell enlargement and attening, formation of senescence-associated heterochromatic foci (SAHF), formation of DNA damage foci (DNA-SCARS), activation of senescence-associated βgalactosidase (SA-β-gal), and increased expression of cell cycle inhibitors p16 and p21. 6 Senescent cells exhibit a phenomenon in which various in ammatory cytokines and various physiologically active substances such as matrix metalloproteases and growth factors (SASP: senescence-associated secretory phenotype) are secreted, and they function to maintain homeostasis such as homeostasis in wound healing. [7][8][9] On the other hand, this phenomenon causes chronic in ammation in surrounding tissues, and it has been suggested that it is involved in the onset and progression of age-related diseases such as cancer, arteriosclerosis, chronic obstructive pulmonary disease, and Alzheimer's disease. 10 Therefore, it is possible that an increase in senescent cells may be involved in the pathogenesis of periodontal disease, which is a chronic in ammatory disease. However, the relationship between senescence and in ammation in periodontal tissue remains unclear.
In this study, we clari ed the dynamics of senescent cells in gingival tissues using an aged mouse model.
Next, we investigated the induction of senescence in gingival broblasts and the associated expression of in ammation-related genes with the aim of determining the relationship between periodontitis and cellular senescence. 10

Materials And Methods
Cell culture Normal human gingival broblasts (HGFs #PCS-201-018, American Type Culture Collection, Manassas, VA) were cultured in DMEM (#D6429, Sigma-Aldrich, St. Louis, MO) with 10% FBS at 37 °C in a humidi ed atmosphere of 5% CO2. 11 The cells were cultured until 70% con uence on 100-mm diameter culture dishes and then transferred to 6-well at-bottom culture plates to perform the following experiments.
Senescence of HGFs was induced by multiple passages or by stimulation with hydrogen peroxide (H 2 O 2 ). 12

Senescence of HGFs induced by multiple passages
HGFs at densities 2 × 10 5 cells per dish were seeded in 100-mm tissue culture dishes. The HGFs were passaged when the culture reached approximately 60-75% con uency. For SA-β-gal staining, 1,500 broblasts were placed in a 35 × 10 mm dish for each passage. 10 µM of the senolytic drug ABT263 (Navitoclax, Chemscine, CS-0013) was added to nine-generation passage-cultured HGFs and the cells were incubated for 24 h. At the end of culture, total RNA was collected and the expression of p16, p21, IL-1β, and TNF-α was examined by real-time PCR.

Senescence of HGFs induced by hydrogen peroxide
At the 5th passage, HGFs were placed in a 35 × 10 mm dish. On day 0, the cells were stimulated with 20 µM hydrogen peroxide (H 2 O 2 , Fuji lm Wako, Japan). 12 At 5 days after stimulation with 20 µM H 2 O 2 , the cells were harvested for SA-β-gal staining, measurement of SA-β-gal activities, and real-time PCR.

Animals
Male C57/B6N mice at the ages of 10 weeks and 20 months (n = 3 per group) were used. The animals were bred in a strictly monitored air-conditioned clean room and were given standard laboratory pellets and water ad libitum at National Center of Geriatrics and Gerontology. The animals were sacri ced and gingival tissues were collected for real-time PCR and primary gingival broblast culture. All experimental procedures were reviewed and approved by National Center for Geriatrics and Gerontology.
The protocol of this animal study was approved by the Research Facilities Committee for Laboratory Animal Science at National Center of Geriatrics and Gerontology (approval number 30-58) and performed in agreement with the ARRIVE guidelines.
ABC biotin solution (Vectastain ABC kit, Funakoshi Co., Ltd, Tokyo, Japan) and ABC reagent were subsequently used, and the sections were then developed using DAB substrate. 13 Primary mouse gingival broblast culture The oral mucosa around the upper maxillary molars was immediately washed in PBS and transferred to a culture dish. Mouse gingival broblasts (MGFs) were grown in DMEM supplemented with 10% FBS. The medium was changed every other day. The cells were grown to semi-con uence, harvested by trypsinization at 37 °C for 3 min, and then subcultivated with DMEM supplemented with 10% FBS in a new dish. In the experiment, cells that had been passaged twice were used as early-passage broblasts. 14 Measurements of SA-β-gal Cells in which senescence had been induced by multiple passages and stimulation with hydrogen peroxide were assessed by SA-β-gal staining using a senescence detection kit (Bio Vision, CA, USA) following the manufacturer's instructions. Cellular SA-β-gal activity was also evaluated using a 96-well cellular senescence assay kit (Cell Biolabs, San Diego, CA, USA) following the assay protocol. 15 Real-time PCR Total RNA was isolated from gingival tissues and HGFs with Nucleospin RNA (Takara Bio Inc., Shiga, Japan, cat no. U0955C) according to the manufacturer's instructions. The total RNA concentration was corrected to 100 ng/µL with a NanoDrop ™2000 spectrophotometer (Thermo Fisher, Tokyo, Japan). Firststrand cDNA synthesis was carried out using a ReverTra Ace-α-kit (TOYOBO, Osaka, Japan). Real-time PCR was performed with FastStart Essential DNA Green Master (Roche, Mannheim, Germany) according to the manufacturer's protocol. One microgram of RNA was reverse-transcribed using the LightCycler96 System (Roche, Germany). The primer sequences used for each gene are shown in Table 1. The samples were normalized to the housekeeping gene, GAPDH, and the results were reported for each sample relative to the control. The experiments were performed in triplicate for each condition. The value is presented as the fold change between the samples using the ΔΔCt method. 13 Statistical analysis Results of statistical analysis are expressed as means ± SEM. The signi cance of differences between groups was examined using one-way analysis of variance (ANOVA) or two-tailed Student's t-test.

Senescent cells in gingival tissues of aged mice
To investigate the state of cellular senescence in gingival tissues, we collected oral mucosa around the upper maxillary molars from aged mice (20-month-old mice) and we examined the expression levels of aging-related molecules (SA-β-gal, p16, and p21) in the tissues and compared them with those in tissues from young mice. First, we examined the mRNA expression levels of p16 and p21 by real-time PCR. The mRNA expression levels of p16 and p21 were increased in gingival tissues from aged mice compared to those in gingival tissues from young mice (Fig. 1A). Next, to investigate the localization of senescent cells in the tissues, we carried out immunohistochemical staining using an anti-p16 antibody. As shown in Fig. 1B, gingival tissues from aged mice were stained more strongly with the anti-p16 antibody than were gingival tissues from young mice. The staining was particularly strong in the granular layer and basement layer of the epithelium and in submucosal tissue of the aged tissues. On the other hand, in young mice, only the granular layer of gingival tissues was strongly stained. SA-β-gal expression was also increased in cells isolated from gingival tissues of aged mice compared to that in cells from gingival tissues of young mice (Fig. 1C). It is known that the expression and secretion of in ammatory cytokines are increased in senescent cells. 16 Therefore, we compared the expression levels of IL-1β and TNF-α in gingival tissues from aged mice with those in gingival tissues from young mice. The mRNA expression levels of IL-1β and TNF-α were signi cantly increased in gingival tissues from aged mice compared to those in gingival tissues from young mice (Fig. 1D). It is known that the ratio of M1 and M2 macrophages (M1/M2 ratio) is altered in gingival tissue of chronic periodontitis. 17 Therefore, we examined the dynamics of macrophages in the collected mouse gingival tissues. Staining of mouse gingival sections with anti-iNOS, anti-CD206, anti-Iba-1 antibodies revealed that there was a large number of macrophages in gingival tissues from aged mice compared to that in gingival tissues from young mice (Fig. 1E). Next, we analyzed mRNA expression levels of iNOS (M1 marker) and CD206 (M2 marker) in gingival tissues by real-time PCR. The expression level of iNOS mRNA was higher in gingival tissues of aged mice than in gingival tissue of young mice. In contrast, the expression levels of CD206 mRNA were not different in gingival tissues of aged mice and young mice (Fig. 1E). To further investigate the relationship between aging and wound healing, we examined the expression of collagen in the same tissues. The mRNA expression levels of collagen 1a1, collagen 4a1, and collagen 4a2 were markedly reduced in gingival tissues from old mice compared to those in gingival tissue from young mice (Fig. 1F).

Senescence of gingival broblasts in vitro
Next,we investigated the properties of cellular senescence in gingival tissues in vitro. Cells are thought to undergo senescence by reaching the limit of the number of possible divisions. Cellular senescence is also induced by application of oxidative stress. Therefore, senescence properties of gingival tissue-derived cells were examined by long-term subculture of HGFs or by applying oxidative stress to the cells. First, senescence of cultured HGFs was assessed by SA-β-gal staining and measurement of SA-β-gal activity. A signi cant increase in the proportion of SA-β-gal cells was observed after 9 passages of gingival broblasts ( Fig. 2A, B). The proportion of SA-β-gal cells continued to increase until passage 12, after which the cells stopped growing. An increase in SA-β-gal activity was also found in gingival broblasts after 10 passages (Fig. 2C).
Next, gingival broblasts were incubated for 5 days after addition of 20 µM hydrogen peroxide, and the degree of senescence was evaluated by using SA-β-gal staining. A signi cant increase in the proportion of SA-β-gal positive cells was observed in the culture on day 5 after stimulation with 20 µM hydrogen peroxide (Fig. 2D, E). We next examined the expression of p16 and p21, which are aging markers associated with the cell cycle, in long-term sub-cultured and hydrogen peroxide-stimulated gingival broblasts. Signi cant increases in the mRNA expression levels of p16 and p21 were observed in gingival broblasts after 10 and 11 passages (Fig. 2F, G). In addition, signi cant increases in p16 and p21 mRNA expression levels were con rmed in gingival broblasts stimulated with 20 µM hydrogen peroxide (Fig. 2H, I).
Next, we investigated how the expression of in ammatory cytokines IL-1β and TNF-α was altered in gingival broblast cultures with increased senescent cells. The mRNA expression levels of IL-1β and TNFα were signi cantly increased in gingival broblasts passaged 9 times compared to those in cells passaged 3 times (Fig. 3A, B). IL-1β and TNF-α mRNA expression levels were also signi cantly increased in gingival broblasts stimulated with 20 µM hydrogen peroxide for 5 days (Fig. 3C, D).
To elucidate the relationship between cellular senescence and expression of in ammatory cytokines, we analyzed changes in the expression of in ammatory cytokines when senescent cells were eliminated. Brie y, nine-generation passage-cultured HGFs were incubated with 10 µM of ABT263 for 24 h, and then the mRNA expression levels of p16, p21, IL-1β, and TNF-α were examined. Treatment with ABT263 reduced the mRNA expression levels of p16 and p21 in nine-passaged HGF cultures to their expression levels in ve-generation cultures (Fig. 4A, B). Furthermore, the mRNA expression levels of IL-1β and TNF-α, which was increased by nine-passaged cultures, were signi cantly decreased by the addition of ABT263 (Fig. 4C, D). These results suggest that the increased expression of proin ammatory cytokines by longterm passaged HGF culture is related to the increase in senescent cells in the culture.

Discussion
Periodontitis is a disorder for which the incidence increases with aging, and senescence is thought to be one of the risk factors of periodontitis. 3 However, the relationship between senescence of periodontal tissues and pathogenesis of periodontitis has remained unclear. In this study, we analyzed gingival aging using a mouse model and cell culture models. We clari ed that aging increases the number of senescent cells in the gingiva and enhances the expression of in ammatory cytokines, which may easily induce in ammation in periodontal tissue. Furthermore, the expression of those in ammatory cytokines was suppressed by removing senescent cells using a senolytic drug. The results suggested that periodontitis could be prevented and treated by regulating senescent cells in periodontal tissues.
Senescence is an important risk factor for lifestyle-related diseases such as arteriosclerotic diseases and diabetes and dementia. 18 Periodontal disease is also a type of lifestyle-related disease 19 , and its relevance to systemic lifestyle-related diseases has been pointed out. Periodontal disease is also a disease for which incidence increases with aging and thus senescence is therefore to be a risk factor for its onset and progression. Periodontal tissue changes with aging. 20 Aging of periodontal tissues results in signi cant anatomical and histological changes that cause decreased tissue homeostasis leading to faster disease progression and delayed tissue repair. 21,22 It is also possible that resistance to infection decreases due to a decrease in immune function in elderly people, and this might be a reason why periodontal disease is more likely to develop. 3 On the other hand, senescence is also observed in various cells constituting the organs of living organisms, and it has been reported that accumulation of senescent cells contributes to the decrease in function and to in ammation of each organ. 23 Cellular senescence is a state in which somatic cells have stopped dividing and proliferating, and it has been shown that expression of cyclin-dependent kinase inhibitors (CDKIs) such as p16 and p21 is enhanced in cellular senescence. 24,25 In this study, enhanced expression of p16 and p21 was con rmed in gingival tissues of aged mice and in gingival broblasts after multiple passages. These facts indicate that the cell cycle of broblasts constituting gingival tissues stops and the cells become senescent with aging. In addition, the increased expression of p16 and p21 is also caused by oxidative stress such as H 2 O 2 . [26][27][28] In this study, the mRNA expression levels of p16 and p21 in gingival broblasts were increased by stimulation with H 2 O 2 . Reactive oxygen species (ROS) are strongly induced by gram-negative bacteria such as periodontal pathogens. 29 Therefore, sustained induction of ROS in periodontal tissue by periodontopathic plaque may also promote gingival aging. Increased secretion of various in ammatory cytokines, chemokines, and extracellular matrix-degrading enzymes from senescent cells has been observed, and it has been suggested that these factors cause chronic in ammation. 30 In this study, we con rmed that aging-related markers were also expressed in oral mucosal tissues of aged mice and that the expression of in ammatory cytokines such as IL-1β and TNF-α was also increased in the tissues. Expression of in ammatory cytokines was also observed in gingival broblasts in which senescence was induced. Furthermore, their expression was suppressed by removing senescent cells. These results suggest that the expression of in ammatory cytokines is enhanced in relation to the increase in senescent cells. MMP-3 (stromelysin-1), which is one of Matrix metalloproteinases (MMPs), degrades collagen in the basement membrane as well as inducing the synthesis of other MMPs such as MMP-1 and MMP-9 and it potently destroys periodontal tissue. 31 On the other hand, the association between aging of periodontal tissue-derived cells and expression of MMP-3 is unclear. In this study, the expression levels of MMP-3 mRNA was also enhanced in aged gingival broblast cultures (supplemental data # 1). The enhanced expression of MMP-3 in senescent broblasts might be involved in the exacerbation of periodontitis.
The mRNA induction of in ammatory cytokines revealed in this study may cause SASP. It is also possible that the SASP factor acts on broblasts around senescent cells, leading to secondary induction of in ammatory cytokines and increasing their expression levels. These results suggest that the increased number of senescent cells in gingival tissue may form a condition that makes periodontal tissue more prone to in ammation by continuously producing in ammatory mediators. In this study, we also clari ed that there was an increase in M1 macrophages in gingival tissues of aged mice along with an increase in total macrophages (Fig. 1E). Macrophages fall into the classical M1 and alternative M2 categories. M1 macrophages release various proin ammatory cytokines and are involved in enhancing the in ammatory response. On the other hand, M2 macrophages are thought to be involved in convergence of in ammation. In addition, changes in the balance of M1/M2 macrophages have been suggested to be involved in the development and progression of in ammation. 32 Therefore, the increase in proin ammatory cytokines and M1 macrophages in the gingiva may be a mechanism for exacerbating in ammation and making it chronic. We speculate that this phenomenon may be the role of aging as a risk factor for periodontitis. In the future, we will con rm this phenomenon by conducting infection experiments with periodontopathic bacteria using aged mice.
The possibility of suppressing the onset of arteriosclerosis, dementia, and cancer by removing senescent cells has been revealed in a mouse model. [33][34][35][36] Reagents that induce selective cell death of senescent cells (senolytic drugs) have been reported. 37,38 ABT-263 is one of them and has been shown to induce cell death such as death of aging human fetal broblasts (MEFs). 39 Therefore, we examined how the expression of in ammatory cytokines was changed by adding ABT-263 to gingival broblasts in which senescence was induced. ABT-263 attenuated the expression of hydrogen peroxide-induced cellular senescence markers and decreased the mRNA expression of IL-1β and TNF-α in the cultures. Therefore, it was thought that the increase in the expression of in ammatory cytokines by the addition of hydrogen peroxide to the cultures was due to an increase in senescent cells. In the future, it may be possible to prevent periodontal disease with senolytic drugs.

Conclusion
In this study, we con rmed that aging and oxidative stress increase senescent cells in mouse gingival tissues and gingival broblast cultures. The increase in senescent cells in the gingiva is caused sustained expression of in ammatory mediators and may contribute to the onset and chronicity of periodontitis.

Declarations Author Contributions
All authors have made substantial contributions to conception and design of the study. MF, MK, JW and MW have been involved in data collection and data analysis. MF, Ka M, YS, YA and Ke M have been involved in data interpretation, drafting the manuscript and revising it critically and have given nal approval of the version to be published.