Analysis of senescence in gingival tissues and gingival fibroblast cultures

Abstract Objective To determine senescence‐associated changes in the gingival tissues of aged mice and gingival fibroblast cultures. Materials and Methods The production of senescence‐associated β‐galactosidase (SA‐β‐gal) and mRNA expression of p16, p21, interleukin (IL)‐1β, and tumor necrosis factor α (TNF‐α) were evaluated in gingival tissues, gingival fibroblasts of 10‐ and 20‐month‐old C57BL/6NCrl mice, and multiple‐passaged and hydrogen peroxide‐stimulated human gingival fibroblasts (HGFs). Changes in molecular expression in HGF cultures due to senescent cell elimination by the senolytic drug ABT‐263 (Navitoclax) were analyzed. Results Compared to 10‐week‐old mice, the 20‐month‐old mice had higher numbers of M1 macrophages. The proportion of cells expressing SA‐β‐gal were also higher in 20‐ month‐old mice than in 10‐week‐old‐mice. Gingival fibroblasts in 20‐month‐old mice expressed less collagen 1a1, collagen 4a1, and collagen 4a2 mRNA than those in 10‐week‐old mice. Compared to control cells, H2O2 treated HGF cells expressed higher levels of SA‐β‐gal and p16, p21, IL‐1β, and TNF‐α. Furthermore, ABT‐263 suppressed HGF cell expression of cytokines after senescence induction. Conclusions Senescence‐associated changes were observed in the gingival tissues of aged mice and HGF cultures. In addition, the potential of senolytic drugs to modify aging‐related changes in the gingiva was shown.

such as chronic periodontitis, and the incidence of periodontitis has been shown to increase with age (Darveau, 2010). Cellular senescence (Coppe et al., 2010) not only affects an individual's aging process but may also be involved in the development of age-related diseases. It is a phenomenon (Hayflick, 1965) of irreversible cell cycle arrest caused by telomere shortening, oncogene and tumor suppressor gene regulation, and radiation and reactive oxygen species-induced stresses. Senescent cells share common characteristics, including cell cycle constancy, cell enlargement and flattening, formation of senescence-associated heterochromatic foci, formation of DNA damage foci (DNA-SCARS), activation of senescenceassociated β-galactosidase (SA-β-gal), and increased expression of cell cycle inhibitors p16 and p21 (Kuilman et al., 2010). Senescent cells exhibit a phenomenon in which various inflammatory cytokines and physiologically active substances, such as matrix metalloproteases and growth factors (senescence-associated secretory phenotype [SASP]) are secreted, and they function to maintain homeostasis within the tissue microenvironment, such as in wound healing (Acosta et al., 2008;Kuilman et al., 2008;Wajapeyee et al., 2008). In contrast, this phenomenon causes chronic inflammation in the surrounding tissues, with suggested involvement in the onset and progression of age-related diseases, such as cancer, arteriosclerosis, chronic obstructive pulmonary disease, and Alzheimer's disease (Watanabe et al., 2017). There have been reports on the use of hydrogen peroxide (H 2 O 2 ) to induce gingival fibroblast senescence (Kiyoshima et al., 2012;Xia et al., 2017) and the effects of senescence of alveolar bone cells on periodontal disease Aquino-Martinez, Rowsey, et al., 2020). Some researchers have also evaluated periodontal ligament cells from donors of different ages (Wu et al., 2015). On the other hand, conventional age-related changes in periodontal tissues have been analyzed morphologically (Van der Velden, 1984). In the elderly, the oral mucosa becomes atrophic. The epithelial tissue becomes thinner, the connective tissue loses its elasticity, and the number of capillaries decreases, resulting in a reduced blood supply. Clinically, the gingiva recedes and becomes less elastic. In addition, a decrease in keratinization and in the number of fibroblasts has been observed in gingival tissue with age (Andreescu et al., 2013). On the other hand, age-related changes in gingival tissue have not been fully analyzed from the molecular and cellular biological perspectives. Therefore, in this study, we examined the molecular dynamics of senescence-related changes in gingival tissues using an aged mouse model and the expression of senescence-and inflammation-related genes in gingival fibroblast cultures. In addition, the possibility of controlling gingival senescence using a senolytic drug was investigated in gingival fibroblast cultures.

| Senescence of HGFs induced by multiple passages
HGFs at a density of 2 × 10 5 cells per dish were seeded in 100-mm tissue culture dishes. HGFs were passaged when the culture reached approximately 60%-75% confluency. For SA-β-gal staining, 1500 fibroblasts were placed in a 35 × 10 mm dish for each passage; 10 μM of the senolytic drug ChemScene, was added to nine-generation passage-cultured HGFs, and the cells were incubated for 24 h. At the end of the incubation period, total RNA was collected, and the expression of p16, p21, interleukin (IL)-1β, and tumor necrosis factor α (TNF-α) was examined by real-time polymerase chain reaction (PCR).

| Senescence of HGFs induced by H 2 O 2
At the fifth passage, HGFs were placed in a 35 × 10 mm dish. On Day 0, the cells were stimulated with 20 μM H 2 O 2 (Fujifilm Wako, Osaka, Japan) (Kiyoshima et al., 2012). Five days after stimulation with H 2 O 2 , the cells were harvested for SA-β-gal staining and assessed for SA-βgal activity, and real-time PCR was performed.

| Animals
Male C57/B6N mice aged 10 weeks and 20 months (n = 18 per group) were used. The animals were bred in a strictly monitored airconditioned clean room and were given standard laboratory pellets and water ad libitum at the National Center for Geriatrics and Gerontology. The animals were sacrificed, and gingival tissues were collected for real-time PCR and primary gingival fibroblast culture.
All experimental procedures were reviewed and approved by the National Center for Geriatrics and Gerontology.
The protocol of this animal study was approved by the Research Facilities Committee for Laboratory Animal Science at the National Center for Geriatrics and Gerontology (approval number 30-58) and the study was carried out in accordance with the current version of the Act on Welfare and Management of Animals (1973).

| Primary mouse gingival fibroblast culture
The oral mucosa around the upper maxillary molars was immediately washed with PBS and transferred to a culture dish. Mouse gingival fibroblasts were grown in DMEM supplemented with 10% FBS. The medium was changed every other day. The cells were grown to semiconfluence, harvested by trypsinization at 37°C for 3 min, and then subcultivated with DMEM supplemented with 10% FBS in a new dish. Cells that had been passaged twice were used as early passage fibroblasts (Iwayama et al., 2012).

| Measurement of SA-β-gal
Cells in which senescence was induced by multiple passages and stimulation by H 2 O 2 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 (Yi et al., 2017). Positively stained cells were counted under a x20 magnification microscope in three random fields for each experimental condition (Dimri et al., 1995).

| Real-time PCR
Total RNA was isolated from gingival tissues and HGFs using 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 using the NanoDrop™ 2000 spectrophotometer (Thermo Fisher, Tokyo, Japan). First-strand cDNA synthesis was conducted using a ReverTra Ace-α kit (Toyobo, Osaka, Japan). Real-time PCR was performed using FastStart Essential DNA Green Master (Roche, Mannheim, Germany) according to the manufacturer's protocol. One microgram of RNA was reversetranscribed using the LightCycler ® 96 System (Roche, Germany); the primer sequences are listed in Table 1.

| Statistical analyses
All values are presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) and two-way ANOVA were used to evaluate the differences between groups.
When significant effects were detected, subsequent post hoc analysis was performed using Tukey's post hoc test, where p < .05 was considered significant. All statistical analyses were performed using EZR (Kanda, 2013) for the R software. This is a modified version of the R commander designed to add statistical functions and is frequently used in biostatistics.

| Senescence-associated changes in gingival tissues of aged mice
To investigate the senescence-associated changes in gingival tissues, we collected oral mucosa around the upper maxillary molars from aged mice (20-month-old mice) and examined the expression 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 of p16 and p21 using real-time PCR.
The mRNA expression of p16 and p21 was higher in the gingival tissues of aged mice than in the gingival tissues of young mice ( Figure 1a).
SA-β-gal expression was also greater in the gingival tissues of aged mice than in those of young mice (Figure 1b). It is known that the expression and secretion of inflammatory mediators are increased in senescent cells (Rodier & Campisi, 2011). Therefore, we compared the expression levels of IL-1β, TNF-α, and iNOS in

| Senescence of gingival fibroblasts in vitro
We

| DISCUSSION
The incidence of periodontitis increases with aging, and senescence is thought to be a risk factor for periodontitis (Ebersole et al., 2016). However, the relationship between senescence of periodontal tissues and the pathogenesis of periodontitis remains unclear. Conventional age-related changes in periodontal tissues have been analyzed morphologically and histologically. However, age-related changes in gingival tissue have not been fully examined from the molecular and cellular biological perspectives.
In this study, we examined the expression of age-related and inflammation-related molecules using gingiva from aged mice and gingival fibroblast cultures. Expression of senescence-associated molecules, mRNA expression of inflammatory cytokines, and the number of M1 macrophages were higher, while mRNA expression of collagen in the gingiva was lower in aged mice than in young mice. In addition, in HGF culture systems that were passaged multiple times or were stimulated with H 2 O 2 , increased mRNA expression of aging cell-related molecules and inflammatory cytokines and decreased mRNA expression of collagen were observed, similar to the results obtained in vivo. These results suggest that in the gingiva, the expression of inflammatory molecules increases with an increase in senescent cells.
Senescence is an important risk factor for lifestyle-related diseases, such as arteriosclerotic diseases, diabetes, and dementia (Ferencz & Gerritsen, 2015). Periodontal disease is also a lifestylerelated disease (Genco & Borgnakke, 2013), and its relevance to systemic lifestyle-related diseases has been reported. Periodontal tissue changes with aging ( Van der Velden, 1984). The aging of periodontal tissues results in significant anatomical and histological changes that cause decreased tissue homeostasis, leading to faster disease progression and delayed tissue repair (Tsalikis, 2010;van der Velden, 1991). It is also possible that resistance to infection decreases due to a decrease in immune function in elderly people, increasing their susceptibility to periodontal disease (Ebersole et al., 2016). Further, the accumulation of senescent cells reportedly contributes to inflammation and a decrease in the function of each organ (Ovadya et al., 2018), and it has been reported that the accumulation of senescent cells contributes to an increase in inflammation, thereby reducing the function of each organ (Ovadya et al., 2018). Cellular senescence is a state in which the division and proliferation of somatic cells stop and the expression of cyclindependent kinase inhibitors, such as p16 and p21, is enhanced (Baker et al., 2011(Baker et al., , 2016. In this study, enhanced expression of p16 and p21 was confirmed in the gingival tissues of aged mice and gingival fibroblasts after multiple passages. This indicates that the cell cycle of fibroblasts constituting gingival tissues stops and the cells become senescent with aging. Increased expression of p16 and p21 also results from oxidative stress, such as that induced by H 2 O 2 in this study (Chen et al., 2019;Macip et al., 2002;Tabasso et al., 2019).
Therefore, sustained induction of ROS in periodontal tissue by periodontopathic plaque may also promote gingival aging. Increased secretion of various inflammatory cytokines, chemokines, and extracellular matrix-degrading enzymes from senescent cells has been observed, and it has been suggested that these factors cause chronic inflammation (He & Sharpless, 2017). In this study, we confirmed that aging-related markers were expressed in gingival tissues of aged mice and that the expression of inflammatory cytokines, such as IL-1β and TNF-α, was also greater in their tissues.
In this study, it was confirmed that the expression of collagen 4 was attenuated together with collagen 1 in the gingiva of aged mice. other MMPs, such as MMP-1 and MMP-9, which can potently destroy periodontal tissue (Sorsa et al., 2004). In contrast, the association between the aging of periodontal tissue-derived cells and the expression of MMP-3 is unclear. In this study, the mRNA expression of MMP-3 mRNA was also increased in aged gingival fibroblast cultures (Figure 5c and progression of inflammation (Sica & Mantovani, 2012). In this study, the total number of macrophages (expression level of Iba-1 mRNA) was significantly higher in the gingival tissues of aged mice than in the gingival tissues of young mice. However, when the M1 and M2 phenotypes were examined, it became clear that both M1 and M2 macrophages were increased in the periodontal ligaments of aged mice. In this study, the function of macrophages in the periodontal tissues of aged mice could not be investigated, and the significance of the findings is unclear. The increase in M1/M2 macrophages in aged periodontal tissues may be associated with reduced infectious immunity and enhanced inflammatory response in aged animals. We need to examine this point in the future. M1-M2 macrophage distinction is too simplistic and barely discernible in vivo (Jablonski et al., 2015). We also speculate that this phenomenon might be due to aging as a risk factor for periodontitis. However, based on the results of this study, it is impossible to conclude the causal relationship between age-related changes in the gingiva and the exacerbation of gingival inflammation, and the development of chronic periodontitis. In the future, we will confirm this relationship by conducting infection experiments with periodontopathic bacteria in aged mice.
The possibility of suppressing the onset of arteriosclerosis, dementia, and cancer by eliminating senescent cells has been demonstrated in mouse models (Bussian et al., 2018;Kovacovicova et al., 2018;Matjusaitis et al., 2016;Zhang et al., 2019). Reagents that induce selective cell death of senescent cells (senolytic drugs) have been reported (Kirkland et al., 2017;Zhu et al., 2015). ABT-263 is one of them and has been shown to induce cell death, such as that of aging human fetal fibroblasts (Chang et al., 2016). Therefore, we examined the change in the expression of inflammatory cytokines following the addition of ABT-263 to gingival fibroblasts in which senescence was induced.
ABT-263 attenuated the expression of H 2 O 2 -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 inflammatory cytokines by the addition of H 2 O 2 to the cultures was due to an increase in senescent cells. Since the elimination of senescent cells is an entirely new therapeutic paradigm, the application of senolytic drugs for human intervention must be critical enough to render currently available treatments ineffective (Kirkland & Tchkonia, 2020). In the future, we would like to examine the effectiveness of senolytic drugs in treating gingival tissue.

| CONCLUSION
In this study, we characterized the expression of aging and inflammation-related molecules in the gingiva of aging mice and HGF cultures. In the future, clarifying the causal relationship between these changes and oral mucosal diseases, such as periodontitis, can contribute to the possibility of using senolytic drugs for oral mucosal diseases.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data sets used and/or analyzed during this study are available from the corresponding authors on reasonable request.

ETHICS STATEMENT
All experimental procedures were reviewed and approved by the National Center for Geriatrics and Gerontology. The protocol of the