Relationship between aging and periodontal disease severity in gauge-raised cynomolgus monkeys (Macaca fascicularis)

Abstract

The study aimed to evaluate the periodontal disease status in different age groups and clarify the relationship between aging and the severity of periodontal disease. The test animals were cynomolgus monkeys that were born and raised at Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition. The participants were divided into three groups: young (5–10 years old), middle (10–19 years old), and old (≥20 years old). The Plaque Index (PLI), Gingival Index (GI), Probing pocket depth (PPD), and Bleeding on probing (BOP) were used for the periodontal examination. Representative teeth were also examined. Polymerase chain reaction (PCR) was used to identify Porphyromonas macacae in dental plaque. Multiple comparisons and regression analyses were used to analyze the relationship between each age group and each oral examination index. Statistically significant differences were found between the age groups and periodontal examination index. Multiple regression analysis revealed that age was strongly correlated with each oral examination index. Based on these results, oral examinations of cynomolgus monkeys kept in the same environment confirmed an association between aging and periodontal disease severity. Monkeys at this facility are expected to serve as new experimental models for elucidating the mechanisms underlying the progression of age-related periodontal disease.

Introduction

Periodontal disease is characterized by the inflammatory destruction of periodontal tissue caused by a host immune response to oral bacterial infection [1]. Plaque formation on the tooth surface causes periodontal disease. Periodontal disease progression is affected by genetic factors; congenital factors such as age, sex, and race; environmental and acquired factors such as smoking, stress, diabetes, obesity, regular drug use, and HIV infection [2]. The progression of periodontal disease was assessed using the probing depth index, which measures the depth of the periodontal pockets from the gingival margin and indicates the degree of periodontal tissue destruction. According to the 2016 Survey of Dental Diseases, the prevalence of periodontal disease increases with age, with 33.1% prevalence among those aged 30–34 years and 57.9% among those aged 60–64 years [3]. Periodontal disease is thought to occur when the immune response to bacteria is modified by environmental and acquired factors [4,5,6,7], while the mechanisms of the relationship between aging and the onset and severity of periodontal disease have not been fully elucidated, it is difficult to clarify the mechanism of periodontal disease progression with aging in human studies due to the influence of confounding factors. Owing to their phylogenetic proximity to humans [8], non-human primates have been examined in many fields as models or in research on cardiovascular, endocrine, and metabolic diseases. Non-human primates are very similar to humans in terms of clinical findings of disease [9]. Studies on nonhuman primates have reported an association between metabolic syndrome and periodontal disease [10]. Although the levels of systemic inflammatory mediators associated with gingival inflammation and periodontal tissue destruction are significantly elevated in adults and older animals, the levels of serum antibodies reactive to periodontal pathogens are less than 50% of those of adult animals in young animals [11]. Furthermore, increase in inflammatory cytokines and antibody titers against periodontal pathogens have been reported in non-human primates, as in humans [11]. When non-human primates with naturally occurring gingivitis were divided into high and low inflammatory groups, differences in inflammatory mediators and subgingival flora were observed; however, these differences were masked in experimental periodontitis induced by ligation [12].

The association of age-related, naturally occurring periodontal diseases in non-human primates, rearing a uniform environment has not been reported extensively. In the present study, we examined the relationship between aging and the progression of periodontal disease in cynomolgus monkeys bred and raised at Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition (TPRC), where the rearing environment is standardized.

Materials and Methods

Animals

A total of 148 monkeys, ranging in age from 5 years 1 month to 31 years 6 month (male: female: 43:105), reference Fig. 1, bred and raised in the TPRC were used in the study. The weight was 3,826.3 ± 985.4 g. The rearing environment was room temperature 25 ± 3°C, humidity 60 ± 5%, ventilation frequency 10 times/h, and light/dark lighting at 12 h intervals. The animals were monitored daily for health and menstruation and were fed 75–100 g of solid feed (CMK-2, CLEA Japan, Tokyo, Japan) and 100 g of apples per day. Water was continuously supplied using an automatic watering system. The composition of the solid diet was 20% protein, 5% fat, and 4% fiber.

Fig. 1.

Breakdown of the number and age of male and female monkeys surveyed.

Health observations such as weight measurement and examination of blood samples were conducted every two years under general anesthesia by a veterinarian. Intraoral and dental examinations were conducted in conjunction with health observations. No interventions, such as oral cleaning, were performed.

This study was approved by the Animal Ethics Committee of Tsukuba Primate Research Center, National Institutes of Biomedical Research and Innovation, Health and Nutrition (approval no. DS29-44R1). Additionally, the investigators attended a training course on animal ethics and biosafety before starting the study.

Intraoral observation

Intraoral observation was performed under general anesthesia via intramuscular administration of ketamine. Multiple individuals performed the examinations. Visual examination of the oral cavity and photography of the frontal and lateral views were performed by a periodontist and two dentists holding the animals’ heads steady.

Dental examination

The periodontal examination sites included six teeth (maxillary right first molar, maxillary left central incisor, maxillary left first premolar, mandibular left first molar, mandibular right central incisor, and maxillary right first premolar) based on the method described by Ramfjord [13]. If the subject tooth was missing, the tooth distal to it was examined using the method of Marthaler et al. [14]. The Plaque Index: Löe and Silness’s mean Plaque Index (PLI) [15]. The Gingival Index: Löe and Silness’s mean Gingival Index (GI) [16] . Probing pocket depth (PPD) for periodontal tissue destruction by periodontitis and Bleeding on probing: Ainamo and Bay’s Bleeding on probing (BOP) rate for periodontal disease activity was used [17]. A disposable probe (CP12; Sakurai, Aichi, Japan) was used to measure the PPD. A four-point method was used for these four indices and only one participant conducted the measurements. The PLI, GI, PPD, and BOP values were measured in that order. The GI was determined by checking the plaque on the margin of the gingiva for bacteriological examination, and BOP was determined by bleeding from the periodontal pocket after the PPD was measured. The following method was used to calculate each oral examination value: the PLI was calculated by dividing the representative value by the number of tooth surfaces where one tooth had the maximum total PLI score. For GI, the representative value was the tooth with the largest total GI score divided by the number of tooth surfaces. PPD is the representative value of the deepest value among the six teeth and is expressed in millimeters. The BOP rate was calculated by dividing the sum of the bleeding areas of the six subject teeth by the number of tooth surfaces to be examined and dividing it by the number of teeth to be examined and was expressed as a percentage.

Microbial examination

Specimens were collected from the supragingival and subgingival plaques of the subjects’ teeth using sterile swabs and paper points [18]. DNA was extracted from the bacteria in the plaque using a DNA extraction kit (Isoplant, Nippon Gene, Tokyo, Japan), suspended in TE buffer (50 mM Tris HCL and 1 mM EDTA, pH 7.6), and used for polymerase chain reaction (PCR). PCR primers were designed and prepared based on the 16S rRNA sequences of Porphyromonas macacae ATCC 49407 (P. macacae). The sequence is 5′- TGG GTT TAA AGG GTG CGT AG −3′ and 5′- GCT TTC GCT CAG GAG CAT AC −3′. Gene amplification by the polymerase chain reaction (PCR) method was performed at 94°C for 30 s for denaturation, 58°C for 30 s for annealing, and 72°C for 60 s for extension in 30 cycles. The amplified DNA fragments were separated by 1.5% agarose gel electrophoresis using Smart Ladder (Nippon Gene) as a molecular weight marker, and the gels were placed on a UV irradiation system and photographed to detect the amplified DNA fragments. PCR products were detected at 301 bp, which is the size of PCR-amplified products reported to be present in periodontal pockets of rhesus monkeys [19,20,21,22] . The detection rate was calculated by dividing the number of PCR-positive individuals by the number of individuals examined and was calculated for the total number of individuals and each age group.

Statistical analysis

The clinical examination data were divided into three age groups: young (5–10 years old, n=55, male:female=19:36), middle (10–19 years old, n=52, male:female=17:35), and old (≥20 years old, n=41, male:female=7:34). The following statistical methods were used to analyze the data: After calculating the median and interquartile range of the data for each of the three groups, normality was confirmed using the Kolmogorov-Smirnov test. The Kruskal-Wallis test was used to compare the results of each oral examination between the groups. A multiple comparison test with the Bonferroni correction was subsequently performed. A chi-square test was also performed to compare the PCR detection rates between the groups. The objective variable was each oral examination value, and the explanatory variables were age (continuous variable) and sex (transformed into categorical factors of male: 1 and female: 0). The explanatory variables were age (continuous variable), sex (transformed into a categorical factor of male: 1, female: 0), and PCR positivity (transformed into a categorical factor of positive: 1, negative: 0) (PCR hereafter). Multiple regression analysis was performed for PLI, GI, and PPD, and logistic regression analysis was performed for the BOP rate. EZR ver1.37 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) was used, and the significance level was set to less than 5%[23]. The graphs were created using Excel 2016 (Microsoft Japan Co., Ltd., Tokyo, Japan).

Results

Oral findings

No caries were observed in any age group. Figure 2 shows the representative intraoral photographs of each of the three groups from top to bottom: young group (5–10 years old, n=55), middle group (10–19 years old, n=52), and old group (≥20 years old, n=41). As characteristics of the intraoral findings, from the young group to the old group, a large amount of plaque, deposition of pigments and calculus, redness and swelling due to gingival inflammation, and significant attrition were observed with aging. Gingival recession progressed in the old group, and large amounts of calculus deposition and gingival inflammation were observed. There was one sample in which the adjacent tooth was measured because it was missing in the old group.

Fig. 2.

Intraoral Photograph. The above and below gingiva shows the intraoral photographs of young (A–C), middle (D–F), and old (G–I). As the age group increases, staining and plaque deposition are observed on the teeth. The peri-gingival area was erythematous and swollen, and gingival retraction was observed compared to that of the young patients.

Table 1 shows the results of the Kruskal-Wallis test analysis of the measurement results of the oral examinations (PLI, GI, PPD, and BOP rate). The values of each oral examination increased significantly with aging. The results of the PLI statistical analysis for each age group are shown in Fig. 3 PLI increased with age, and statistically significant differences were found between young and middle (P=2.7 × 10⁻²), middle and old (P=1.0 × 10⁻⁵), and young and old groups (P=3.1 × 10⁻¹⁰). The median values for the young, middle and old groups are 1.00, 1.25 and 1.75, respectively. Figure 4 shows the results of the statistical analysis of GI for the age groups. The old group showed an increase in GI compared with the young and middle groups. Statistically significant differences were found between the young and old groups (P=4.3 × 10⁻¹⁰) and between the middle and old groups (P=7.3 × 10⁻⁷) except for the young and middle groups. The median values for the young, middle and old groups are 0.50, 1.25 and 0.50, respectively. Figure 5 shows the results of PPD statistical analyses for each age group. PPD increased with age. Statistically significant differences were observed among all age groups. Statistically significant differences were observed between young and middle (P=5.7 × 10⁻⁴), middle and old (P=4.2 × 10⁻⁹), and young and old (P=5.9 × 10⁻¹³).The median values for the young, middle and old groups are 1, 2 and 3mm, respectively. Figure 6 shows the results of the statistical analysis of BOP rate for each age group. The BOP rate increases with age. Statistically significant differences were observed between young and middle (P=2.3 × 10⁻⁴), middle and old (P=1.3 × 10⁻³), and young and old (P=5.5 × 10⁻¹⁰). The median values for the young, middle and old groups are 0, 0 and 13%, respectively. Supplementary figures show a one-to-one correlation between age and periodontal examination (PLI, GI, PPD and BOP rate) for each individual, not taking into account PCR results and sex (Supplementary Figs. 1–4).

Table 1. Median values of PLI, GI, PPD, and BOP rate in each group^a).

	young (n=55)	middle (n=52)	old (n=41)
PLI	1.00 (0.23–1.25)	1.25 (0.75–1.75)	1.75 (1.50–2.50)
GI	0.50 (0.25–0.63)	0.50 (0.75–1.75)	1.25 (0.75–1.75)
PPD (mm)	1 (1–2)	2 (2–2)	3 (3–4)
BOP rate (%)	0 (0–0)	0 (0–4)	13 (0–21)

^a)Interquartiel ranges are shown in brackets.

Fig. 3.

PLI (Mann-Whitney U test with the Bonferroni correction) for each group are shown in a box-and-whisker diagram. *: P<0.05, **: P<0.001. Error bars indicate maximum and minimum values, and the center line indicates the median. Statistically significant differences were found between young and middle group (P<0.05), middle and old group (P<0.001), and young and old group (P<0.001).

Fig. 4.

GI in each group (Mann-Whitney U test with the Bonferroni correction) is shown in a box-and-whisker diagram. *: P<0.05, **: P<0.001. Error bars indicate maximum and minimum values, and the center line indicates the median. The dots in the error bars indicate values outside the interquartile range. There were statistically significant differences between the young and middle groups (P<0.05), the middle and old groups (P<0.001), and the young and old groups (P<0.001).

Fig. 5.

PPD in each group (Mann-Whitney U test with the Bonferroni correction) is shown in a box-and-whisker diagram. **: P<0.001 is shown. Error bars indicate maximum and minimum values, and the center line indicates the median. Dots in the error bars indicate values outside the interquartile range. There were statistically significant differences between the young and middle groups (P<0.05), the middle and old groups (P<0.001), and the young and old groups (P<0.001).

Fig. 6.

The BOP rate for each group (Mann-Whitney U test with the Bonferroni correction) is shown in a box-and-whisker diagram. *: P<0.05, **: P<0.001. Error bars indicate maximum and minimum values, and the center line indicates the median. Statistically significant differences were found between young and middle group (P<0.001), middle and old group (P<0.05), and young and old group (P<0.001).

Analysis of P. macacae in dental plaque

The detection of P. macacae in dental plaque samples using PCR. In total, 75 out of 148 individuals tested were positive (51%). In the young group, 22 out of 55 individuals tested were positive (40%). In the middle group, 27 of 52 animals tested were positive (51%). In the old group, 27 of 41 animals tested were positive (66%). In each age group, there was no statistically significant difference between the age group and bacterial positivity (chi-square test, P=0.81).

Multivariate analysis

Table 2 presents the results of the multiple regression analysis. In the PLI, only age showed a statistically significant difference, and the standardized partial regression coefficient (β)=0.584 (P=2.3 × 10⁻¹⁴), showing that PLI was correlated with age. For GI, there was a correlation between age and the presence of bacteria. The β values for age and bacteria were β=0.569 (P=1.4 × 10⁻¹⁴) and β=0.241 (P=3.2 × 10⁻⁴), respectively. GI showed a correlation between age and bacteria. For PPD, correlations were found between all factors, including age, sex, and bacteria. The β for age=0.667 (P=5.5 × 10⁻²⁰), for gender=−0.127 (P=4.3 × 10⁻²), and for PCR=0.124 (P=4.5 × 10⁻²). PPD was correlated with age, sex, and the presence of bacteria. The results of the logistic regression analysis are presented in Table 3. The odds ratio (OR) of the BOP rate to age was 1.01 (P=1.1 × 10⁻⁸), indicating that the BOP rate statistically correlated with age.

Table 2. Multiple regression analysis results.

Objict variable	Dependent variale	Paritial regression coefficient	Standard deviation	Standardized paritial regression coefficient	95% Confidence Interval		P	R2
					Upper	Lower
PLI	Age	0.0043	0.0050	0.5840	0.0030	0.0053	2.3×10−14	0.3240
	Sex	–0.1690	0.1060	0.1090	–0.3800	0.0041	nsa)
	PCR	0.0928	0.0096	0.0650	–0.1000	0.2830	nsa)
GI	Age	0.0035	0.0004	0.5690	0.0030	0.0040	1.4×10−14	0.3710
	Sex	–0.1682	0.0852	0.1310	–0.3400	0.0000	nsa)
	PCR	0.2845	0.0077	0.2410	0.1390	0.4370	3.2×10−4
PPD	Age	0.0016	0.0001	0.6670	0.0010	0.0020	5.5×10−20	0.4430
	Sex	–0.0629	0.0308	0.1270	–0.1200	–0.0020	4.3×10−2
	PCR	0.0566	0.0279	0.1250	0.0010	0.1120	4.5×10−2

^a)ns: not significant.

Table 3. Logistic regression analysis results.

Objict variable	Dependent variale	Odds ratio	SD	95%confidence interval		P
				Upper	Lower
BOP rate	Age	1.01	0.00	1.01	1.02	1.1×10−8
	Sex	1.92	0.47	0.77	4.79	nsa)
	PCR	1.01	0.41	0.45	2.23	nsa)

^a)ns: not significant.

Discussion

Experimental periodontitis models include those in which inflammation is induced by ligating silk threads around the teeth, and in which defects are surgically created in the alveolar bone using cutting instruments. Ebersole et al. conducted a study using 20 female cynomolgus monkeys [11] and another study using 16 adult female cynomolgus monkeys [12] to elucidate the pathogenesis of periodontal disease using experimental periodontitis induced by ligating silk threads. These studies offer useful models to examine the tissue responsiveness and systemic effects of inflammation on periodontal tissue. However, unlike the actual pathophysiology of periodontal disease, ligature-induced periodontitis experimentally induced by ligatures is an acute inflammation that immediately disappears when the ligatures are removed. In the surgical periodontal tissue defect model, the gingiva is surgically detached and lifted from the tooth surface to expose the alveolar bone, and a cutting instrument was used to create an alveolar bone defect adjacent to the tooth. This model is useful for studying the effects of interventions and treatment modalities on periodontal tissue healing.

Periodontitis is the destruction of periodontal tissue due to chronic inflammation caused by plaque produced by oral bacteria [1], and epidemiological studies have reported a high incidence of periodontitis in the elderly [3]. However, host and environmental factors, in addition to bacterial factors, affect the onset and severity of periodontitis (a multifactorial disease). It is difficult to clarify the relationship between aging and periodontitis in humans. In this study, we examined the relationship between aging and severity of periodontal disease by evaluating the condition of periodontal tissues in different age groups of cynomolgus monkeys raised under constant environmental conditions from birth. We observed an association between aging and the severity of periodontitis by minimizing the effects of environmental factors, such as stress stimuli and diet, and by using cynomolgus monkeys with naturally occurring periodontitis. To examine the effects of aging and the severity of periodontitis, we divided the subjects into three groups: young (5–10 years), middle (10–19 years), and old (≥20 years) based on the following rationale: a high prevalence of periodontitis in U.S. adults over 30 years of age and the fact that ~50% are affected by periodontitis suggests that the age of onset of the disease is around 30 years. Eke et al. reported that data from the 2009–2012 National Health and Nutrition Examination Survey (NHANES) in the U.S. showed that almost 50% of U.S. adults aged 30 years or older have periodontitis [24]. Furthermore, Saminsky et al. reported that the risk of tooth loss after initial periodontal disease treatment was 3.3 times higher in patients aged 60 years or older than in controls among 50 patients receiving long-term maintenance treatment [25]. Chambrone et al. reported that older age (>60 years) was one of the causes of tooth loss in 120 patients with periodontal disease who received periodontal maintenance for >10 years [26]. Furthermore, we determined the age category with reference to a report that approximately three times the age of non-human primates corresponds to the human age [9]. However, age comparison between monkeys and humans varies greatly depending on the index used; therefore, three times is the benchmark.

Oral examination showed that each parameter worsened with age, and statistically significant differences were observed in the PLI, GI, PPD, and BOP rate (P<0.001). In the comparison of oral examination values and age groups, statistically significant differences were found between the young and old groups (P<0.001) and between the middle and old groups (P<0.001) in GI. However, there were no statistically significant differences between the young and middle groups. In contrast, significant differences in the PLI, PPD, and BOP rate were found between the young and middle groups. The difference in GI, which indicates the degree of inflammation of the marginal gingiva, may be influenced by the amount and duration of plaque formation and calculus deposition. This suggests that the amount and duration of plaque and calculus deposition increased in the old group compared to those in the middle group, which may have affected the GI score. However, the significant differences in PPD and BOP rate between the young and middle groups may be due to the accelerated destruction of periodontal tissues and the progression of inflammation caused by occlusal trauma, in addition to inflammation from a young age. This result is also consistent with the results of Miller et al. in 116 baboons, in which PLI, GI, and PPD increased with age [27]. Of the 148 animals analyzed, 59 were raised using artificial suckling. The nutritional intake during breastfeeding and whether or not a child drinks colostrum may affect the development of periodontal disease.

It is possible that the target individuals in this study included those whose mothers refused to nurse them after drinking colostrum and were forced to use artificial feeding, making it difficult to compare samples with a sufficient number of people of the same age; therefore, a detailed analysis was not possible. This issue should be addressed in future studies.

The reasons for the absence of caries in the individuals examined in this study could be due to the absence of caries-causing bacteria such as Streptococcus mutans and Streptococcus sobrinus or the high acid resistance of the teeth; however, further research is needed on the properties of teeth and oral bacteria of the monkeys.

Nonhuman primates have the same rows and number of teeth as humans; however, there are some differences in their characteristics. Animals in which tooth loss is fatal have longer tooth roots than those of humans, and a tendency toward gingival recession is observed due to the formation of periodontal pockets where chronic periodontitis (loss of tooth and gingival attachment) causes maturation of bacteria in the plaque. This may be a pathological condition that can be described as a combined objective to prevent tooth loss, which can be fatal in monkeys. On the other hand, the root bifurcation position is close to the tooth-gingival attachment area, and gingival recession tends to progress to lesions at the root bifurcation area. Thus, the comparative anatomy of teeth in monkeys and humans leaves room for further study on the validity of the examination site and evaluation method.

Xavier et al. reported the inflammatory response and plaque composition in cynomolgus monkeys with periodontal disease [28]. The inflammatory response in periodontal disease is very similar to that in humans, and the connective tissue is infiltrated with plasma cells, lymphocytes, and neutrophils, which are composed of gram-positive rods and cocci in the supragingival plaque, and anaerobic gram-negative rods in the subgingival plaque. P. macacae, the target of the PCR method used in this study, is a biased anaerobic bacterium reported to have been isolated and purified from the periodontal pockets of rhesus monkeys by Slots et al. [22]. Fournier et al. reported that P. macacae has trypsin-like activity [19]. P. macacae is a periodontopathic bacterium that causes bone resorption during experimental infections in rats. Colombo et al. suggested that Porphyromonas gingivalis (P. g), a pathogenic bacterium in human periodontal disease, may not be as important as a periodontopathic bacterium in monkeys because it was detected equally in periodontally affected and healthy areas in rhesus monkeys used in their experiments [9]. In the present study, the PCR detection rate of P. macacae in each age group were 40% young, 51% middle, and 66% old, which were assumed to be periodontopathic bacteria in monkeys. The detection rate tended to increase with age, but no statistically significant difference was observed. However, multiple regression and logistic regression analyses showed that P. macacae positivity was significantly correlated with GI and PPD. This suggests that P. macacae is associated with gingival inflammation and the loss of gingival attachment to the tooth surface in cynomolgus monkeys. In this study, we conducted a bacteriological evaluation of plaques on the gingival margin in the present study. However, considering that P. macacae is a biased anaerobic bacterium, it is necessary to compare the distribution of the bacterial flora in the subgingival plaque and examine the inflammatory response by histological evaluation.

One limitation of this study is that it was a cross-sectional study. In the future, a longitudinal study should be conducted with a larger number of subjects, a more sex-neutral sample, and radiographic evaluation of hard tissues. In addition, more detailed studies should be conducted, including testing for inflammatory cytokines and aging-related molecules and quantitative PCR methods. It is possible to consider acute and chronic conditions and analyze pathological conditions by obtaining information from the same individual over time.

Furthermore, from a biosafety standpoint, all people entering the animal rooms at the TPRC must wear masks and protective clothing. These biosafety rules may help prevent long-term transmission of caries-causing bacteria from humans to animals. Veterinarian checkups and blood tests were conducted once every two years. However, underlying diseases may have been overlooked. To analyze the relationship between systemic diseases and periodontal disease, it is necessary to analyze the disease itself, as well as a more precise analysis of periodontal disease. The role of the oral environment in systemic health is currently attracting attention because periodontal disease has been implicated in individual health issues, including diabetes, cerebrovascular disease, obesity, and low birth weight. We hope that this study provides insights into the maintenance and management of animal health.

Conclusion

Oral examination of cynomolgus monkeys bred and raised in the same rearing environment revealed that clinical findings of periodontal disease worsened with age. This result is similar to those of previous comparative experiments between young and old individuals using experimental periodontitis. However, it is extremely important that periodontal disease develops spontaneously and progresses even when the influence of environmental factors is excluded, and that cynomolgus monkeys can serve as an animal model of periodontal disease in humans. The use of such animal models will make it possible to analyze detailed mechanisms pertaining to the pathophysiology of aging and periodontal disease and is expected to lead to the development of new preventive and therapeutic methods for periodontal disease in a hyper-aged society.

Conflict of Interest

The authors declare no conflicts of interest associated with this manuscript.