Age‐dependent changes of the mandible bone throughout the lifespan in female F344/N rat

Abstract

Age‐dependent changes of the mandible bone in female F344/N rats, aged 22–1196 days, were analyzed using physiological bone properties and morphology. Bone weight, bone area, bone mineral components, and bone mineral density were assessed using dual‐energy X‐ray absorptiometry. The bone weight, bone area, bone mineral components, and bone mineral density increased rapidly until approximately 150 days of age, increased gradually thereafter, and then stabilized or decreased after 910 days of age. The ratio of bone mineral components to bone weight (bone mineral ratio) increased rapidly until approximately 43 days of age and stabilized thereafter. Size of the mandible, which was measured at 13 points on mandible surface, increased with age, and the rate of change showed a similar pattern to the other parameters. From a principal component analysis on morphometric measurements, principal component 1 (size factor) increased proportionally with age, whereas principal component 2 (shape factor) decreased until approximately 88 days of age and then increased after 365 days of age. As a result, the scatter plots for principal component 1 and principal component 2 were V‐shaped, which indicates that the mandible developed in size, with deformation at younger ages, and recovered its original shape later in life. Our results revealed the occurrence of inflection points at approximately 43, 88, 150, 365, and 910 days of age. Some of these ages corresponded to transition points revealed by the age‐dependent changes of the occlusal mandibular condyle and tooth wear in the same rat.

Introduction

The mandible is a suitable indicator organ that can be used to evaluate the animal's physical status, since it reflects feeding and nutritional conditions in addition to genetic makeup (Cavalli et al. 2015; Nishijima et al. 2017; Wiener & Wiener, 2015). Mandible characteristics include occlusal wear and caries in teeth, and mandible morphology and bone properties. In terms of model animals, morphological examination of the mandible showed substrain differences among F344/DuCrlCrlj, F344/Jcl, and F344/NSlc rats (Tanaka et al. 2006a,b). The mandible is also affected by age and is commonly used as an indicator of age in humans (de Oliveira et al. 2015; Leversha et al. 2016), and it is known that various mandible regions change with age, including the gonial region, antegonial region, condyle, and ramus in humans (Ghosh et al. 2010).

Gerontological and geriatric sciences are becoming more important with the expansion of an aging society, and appropriate animal models must be discussed for research in these fields because the way species age differs. Therefore, it is important to identify specific aging properties of the animals so that experimental results may be extrapolated to humans. F344/N, an inbred strain of rat, is one of the most commonly used model animals in gerontological and geriatric sciences; aging properties in the F344/N rat have been investigated from various aspects, including survival (Tanaka et al. 2000a,b, 2001), respiratory tract characteristics (Yamamoto et al. 2003a,b), and reproduction (Sone et al. 2007; Nishijima et al. 2013). Additionally, we previously revealed age‐dependent changes in the mandible focusing on the periodontal bone (Arai et al. 2005), the condyle (Nishijima et al. 2010), and occlusal tooth wear (Nishijima et al. 2007, 2009) in F344/N rats. However, the effects of aging on other elements of the mandible, such as morphology and bone properties, have not yet been clarified.

The current study was conducted to investigate age‐dependent changes of mandibular morphology and bone properties in female F344/N rats, a widely used model animal in gerontological and geriatric research.

Materials and methods

Seventy‐two female F344/NSlc rats from 22 to 1196 days old (D) were used. The rats from 22 to 180 D were purchased from SLC Japan (Hamamatsu, Japan), and the others were reared at the NILS Aging Farm (Tanaka et al. 2000a,b, 2001).

The rats were euthanized with a terminal dose of anesthesia (pentobarbital sodium), and the whole tissue around the oral cavity was dissected out. The dissected tissue was autoclaved at 121 °C for 10 min. After carefully removing the soft tissue, the mandible was incubated overnight in 0.5% papain (Merck, Germany) solution at 37 °C to digest the remaining attached soft tissue. After rinsing, a dried bone specimen of the mandible was prepared (Tanaka et al. 2006a,b). The mandibles were divided into 10 age groups (22–25, 30, 42–44, 77–92, 129–168, 181–214, 350–377, 726–732, 909–916, and 1087–1196 D). These ages of the rats were chosen to capture the aging properties at particular ages, e.g. 1 year (age group 350–377), to capture the aging pattern of the mandible bone throughout the rat's lifespan.

Incisors were removed before measurements were taken. Each mandible was weighed (BnW, mg) and analyzed to determine bone mineral content (BMC, mg), bone area (AREA, cm²), and bone mineral density (BMD, mg/cm²) using a dual‐energy X‐ray absorptiometry (DEXA) measurement system (DCS‐600EX‐IIIR, Aloka, Tokyo, Japan). A ratio of BMC to BnW (bone mineral ratio, BMR) was calculated (Tanaka et al. 2006a; Tsujio et al. 2009). Since these measurements were performed on mandibles with three molar teeth, the resultant values included both bone and tooth weight as well as calcium (hydroxy) apatite.

The averages of the bone parameters for each age group were calculated and compared using one‐way anova followed by Bonferroni correction. A P < 0.05 was considered significantly different.

The right mandible bones were measured at 10× magnification using a vertical projector with built‐in digital counter and protractor (V‐20B, Nikon, Tokyo, Japan). The digital device provides an accurate size of the bone, and each distance was measured three times and averaged. The measurements were taken at 13 points as illustrated in Fig. 1. Seven of the 13 points (X ₁ to X ₇) were distances from the horizontal axis (i.e. height), and the other six points (X ₈ to X ₁₃) were distances from the vertical axis (i.e. length), in accordance with Festing's method (1979).

Figure 1.

Schematic illustration of the tongue‐side view of a mandible including the 13 measurement points. Distances from the horizontal axis to X ₁–X ₇ points, and distances from the vertical axis to X ₈–X ₁₃ were measured following the previously reported method of Festing (1979).

The one‐way anova followed by Bonferroni correction was used to compare each measured distance among age groups. Rates of change between length and height in each age group were compared using Student's t‐test. Again, P < 0.05 was considered significantly different.

Results were run in a principal component analysis (PCA) using spss Statistics 24.0 (IBM, Armonk, NY, USA).

Results

The mandible bones of younger rats were thin, and their surfaces were clean and smooth. In contrast, mandible bones of older rats were thick with rough surfaces. In older rats, dental caries and periodontitis were observed and some molar teeth were lost from the mandible in some cases. The surfaces of the condylar process were rough and were occasionally accompanied by a depression (Fig. 2).

Figure 2.

Mandibles of (A) a younger (181–214 D group) and (B) an older (1087–1196 D group) rat. Caries (arrow), occlusal tooth wear (arrowheads), periodontitis (double arrowhead) and rough condyle with a depression are observed in the older rat specimen.

BnW increased rapidly between approximately 24 and 150 D and thereafter increased gradually, reaching 354.7 ± 42.2 mg at 1129 D (Fig. 3A). The values tended to vary in the older age groups and decreased in the oldest age group (Fig. 3A,B). Although AREA, BMC, and BMD behaved similarly to BnW, BMD increased more rapidly in the younger age groups (Fig. 3C). The rate of change at different ages varied from parameter to parameter (Fig. 3D). Throughout the rats’ lifetime, the rates of change of BnW and BMC were higher, particularly after 728 D, than those of other parameters (Fig. 3D). The BMR increased rapidly until approximately 43 D and was mostly stable thereafter (Fig. 3).

Figure 3.

Age‐dependent changes of (A) bone weight (BnW) and bone area (AREA), (B) bone mineral content (BMC), (C) bone mineral density (BMD) and bone mineral ratio (BMR), and (D) the rate of change from the youngest group. # P < 0.01, * P < 0.05: values that are significantly different from the neighboring younger group.

The mandible size, i.e. values of X ₁–X ₁₃ (Table 1), increased with age (Fig. 3, AREA); however, the rate of change differed among the measurement sites (Table 1). The rates of change of heights (X ₁–X ₇), especially X ₁, were greater than those of lengths (X ₈–X ₁₃), as shown in Fig. 4.

Table 1.

Aging change of mandible size

Aging groups (days of age)	No. of animals	X 1	X 2	X 3	X 4	X 5	X 6	X 7
Height (horizontal axis)
22–25	4	0.59 ± 0.08	2.91 ± 0.14	4.27 ± 0.17	5.27 ± 0.19	5.81 ± 0.13	6.37 ± 0.08	7.67 ± 0.21
30	3	0.90 ± 0.06**	3.35 ± 0.03	4.80 ± 0.08*	6.12 ± 0.04**	7.06 ± 0.16**	7.73 ± 0.13**	9.28 ± 0.15**
42–44	3	1.14 ± 0.06	3.48 ± 0.06	4.92 ± 0.08	6.51 ± 0.09	7.87 ± 0.16**	8.68 ± 0.12**	10.12 ± 0.19**
77–92	4	1.31 ± 0.03	3.74 ± 0.05	5.30 ± 0.11	7.31 ± 0.09*	9.16 ± 0.16**	10.11 ± 0.23**	11.58 ± 0.18**
129–168	13	1.54 ± 0.07**	4.00 ± 0.07	5.59 ± 0.12	7.85 ± 0.12	9.85 ± 0.23**	10.87 ± 0.3**	12.47 ± 0.3**
181–214	6	1.59 ± 0.03	4.01 ± 0.04	5.61 ± 0.08	7.90 ± 0.06	9.88 ± 0.12	10.93 ± 0.16	12.51 ± 0.14
350–377	9	1.71 ± 0.07	4.24 ± 0.08	5.93 ± 0.10	8.24 ± 0.15	10.21 ± 0.11	11.23 ± 0.14*	12.88 ± 0.14
726–733	10	1.94 ± 0.08**	4.62 ± 0.17**	6.51 ± 0.19**	8.78 ± 0.36	10.58 ± 0.29	11.68 ± 0.22**	13.48 ± 0.25**
909–916	10	2.04 ± 0.14	4.96 ± 0.32*	6.87 ± 0.27**	9.15 ± 0.34	10.91 ± 0.27	11.92 ± 0.30	13.83 ± 0.34
1087–1196	10	2.03 ± 0.17	4.84 ± 0.37	6.85 ± 0.34	9.05 ± 0.45	10.83 ± 0.21	11.92 ± 0.24	13.70 ± 0.25

Aging groups	X 8	X 9	X 10	X 11	X 12	X 13
Length (vertical axis)
22–25	5.87 ± 0.13	13.90 ± 0.29	15.48 ± 0.52	16.07 ± 0.47	17.47 ± 0.49	18.19 ± 0.52
30	6.43 ± 0.12	15.35 ± 0.06**	17.08 ± 0.19**	18.19 ± 0.09**	20.13 ± 0.14**	20.45 ± 0.09**
42–44	6.57 ± 0.10	16.21 ± 0.07	17.74 ± 0.35	19.10 ± 0.04	21.15 ± 0.13	21.18 ± 0.08
77–92	7.24 ± 0.24	18.02 ± 0.37**	19.54 ± 0.28**	20.95 ± 0.39**	23.73 ± 0.48**	23.22 ± 0.41**
129–168	7.62 ± 0.44	18.64 ± 0.47	20.21 ± 0.48	21.67 ± 0.47	24.85 ± 0.44**	24.08 ± 0.46*
181–214	7.49 ± 0.58	18.82 ± 0.6	20.41 ± 0.72	21.84 ± 0.63	24.92 ± 0.66	24.25 ± 0.66
350–377	8.04 ± 0.28	19.26 ± 0.29	20.93 ± 0.32	22.27 ± 0.32	25.51 ± 0.26	24.90 ± 0.26
726–733	8.34 ± 0.18	19.98 ± 0.36**	21.80 ± 0.44**	23.22 ± 0.40**	26.35 ± 0.45**	25.87 ± 0.45**
909–916	8.59 ± 0.37	20.31 ± 0.44	22.12 ± 0.34	23.54 ± 0.35	26.64 ± 0.40	26.41 ± 0.47
1087–1196	8.82 ± 0.54	20.40 ± 0.23	22.17 ± 0.28	23.56 ± 0.18	26.53 ± 0.22	26.38 ± 0.24

Mean ± SD.

**P < 0.01, *P < 0.05: significantly different compared with neighboring younger group.

Figure 4.

Rate of change in size of mandible with age. ^# P < 0.01, *P < 0.05: values that are significantly higher than lengths in the same age group.

When plotted (PC1 on the x‐axis and PC2 on the y‐axis), PC1 was proportional to age, i.e. PC1 increased with age (Fig. 5). However, PC2 showed a V‐shaped trend, i.e. it decreased with age until approximately 150 D and then increased after approximately 365 D to the value calculated for approximately 24–30 D (Fig. 5).

Figure 5.

Scatter plots of PC1 and PC2 showing the mandible morphology at each age. Mean ± SD.

Discussion

The present study demonstrated age‐dependent changes of the mandible bone in terms of physiological bone status and morphological properties in female F344/N rats throughout their lifespan. Furthermore, data from the different analyses corresponded with each other. Since standard deviations of the data were relatively small (except for PC1, especially in elder rats) and a number of significant differences were detected among the aging groups in each parameter, the present results would be reproducible.

Osteoporosis is one of the major geriatric complaints and is characterized by a decrease of BMC and BMD (Nielsen, 2000; von Wowern, 2001). The DEXA measurement calculates BMD by dividing BMC by AREA, thereby reflecting the bone size on the plane surface only, not in depth; thus, bone shape would modify BMD. Since the rat mandible used in the present study is relatively flat because the bilateral halves of the jaw are not fused, we expected the DEXA measurement to be suitable for identifying its BMD. As a result, the rates of change of BMC and BnW with age far exceeded and were not parallel to that of AREA, and the BMR (ratio of the BMC to BnW) was stable. These results indicate that there is a disparity between bone growth and value for AREA, and that thickness of the mandible bone would influence BMD as measured using DEXA. Therefore, excepting BMD, the bone parameters would show the nature of change of the bone properties with age. Furthermore, DEXA measurements of BMD vary widely among bones, even in a single individual (for example, 83.6 mg cm⁻² in the mandible, 56.3 mg cm⁻² in the atlas, 105.5 mg cm⁻² in the lumber vertebrate, and 130.5 mg cm⁻² in the femur). In contrast, BMRs were relatively stable among bones (0.585 in the mandible, 0.492 in the atlas, 0.519 in the lumber vertebrate, and 0.538 in the femur; K. Nishijima and S. Tanaka, unpublished data), and thus BMR may be a valuable parameter for comparing properties of different bones in the same individual or species (Tsujio et al. 2009). Moreover, if a standard BMR per age is established, BnW could be calculated from BMC measured using DEXA and utilized in clinical situations.

In elderly women, severe loss of BMC leading to osteoporosis may be caused by depletion of estrogens associated with the cessation of ovarian functioning, i.e. menopause. The present results showed that the BMC of the mandible was maintained even in old rats. Our previous studies revealed that the cornified cell phase ceased at approximately 16.4 months (circa 500 days) of age in F344/N rats (Sone et al. 2007). Nevertheless, follicles and the corpora lutea were observed in the rat ovary even at 36 months (ca. 1100 days) of age (Nishijima et al. 2013), which indicates that estrogens were retained. This may explain bone parameter maintenance in the old rats investigated in the present study. There appeared to exist three phases in the age‐dependent changes of the mandibular bone parameters and morphology. The first acute increasing phase lasted until approximately 150 D, and the second moderate increasing phase between approximately 150 and 910 D. This was common to BnW, AREA, BMC, BMD, and rate of change of the bone properties and size (Figs 3 and 4). Therefore, there appears to be a major inflection point around 150 D. Since the values decreased between approximately 913 and 1130 D, another inflection point may exist at that age. Additionally, focusing on BMR revealed another inflection point at approximately 43 D, between the acute increasing and stable phases (Fig. 3C).

The principal component analysis (PCA) revealed unique and notable morphological changes of the mandible. The behavior of PC1 correlated closely with those of the bone parameters and size: an acute increase until approximately 88 D, followed by a moderate increase, and finally stabilization or decrease in the oldest rats. In a previous study, by analyzing the relation between the PCs and actual measured values (X ₁–X ₁₃), we estimated that PC1 is a size factor and PC2 a shape factor of the mandible (Tanaka et al. 2006b), which would indicate that PC1 is a factor of mandible size. In contrast, PC2 showed V‐shaped trends, and the value at approximately 913 D was almost equal to that of young (24–30 D) rats. PC2 may be indicative of mandible shape, and mandibles may develop in size with a deformation during the younger phase but recover their shape after 365 D. The lifespan can be divided into several phases including growth, maturation, aging, and senescence, and the primary object of basic gerontology is to associate these phases with the respective physiological status. Our results revealed inflection points at approximately 43, 88, 150, 365, and 910 D, which might be transition points of the aging phases. We have observed various aging profiles in the F344/N rat (Arai et al. 2005; Tsukahara et al. 2005; Nishijima et al. 2007, 2009, 2010, 2013, 2017; Sone et al. 2007), and some of them correlate with the inflection points in our results. During analysis of the mandibular condyle, we found inflection points between 1 and 2 months, at approximately 12 months, and also at 30 months of age (Nishijima et al. 2010). These correspond to 43, 365, and 910 D, respectively, where inflection points were revealed in the present study. Additionally, our results revealed a prominent inflection point at approximately 150 D. Although we did not focus on this age previously, important stages in the rat life cycle may occur around 150 D.

Mendelson & Wong (2012) proposed that aging in the human mandible is characterized by good skeletal structural support in youth, a combination of soft tissue and bony changes in facial aging, and bone loss in specific areas of the facial skeleton. Lipski et al. (2013) reviewed age‐related morphological change of the mandible and discussed the size of the corpus and condyloid process, the angle between the corpus and the ramus, and the position of the canal, mental foramen, and the condyloid process. However, it is not easy to clarify the nature or identify the morphological change of the mandible with age, since it is affected by factors such as dental status (Chole et al. 2013; Ozturk et al. 2013).

Since the F344/N rat is one of the most commonly used model animals in gerontology and geriatrics, our results add to progress in these sciences. Osteological features, especially in the femur, are similar in the rat and human when compared with other fields of research such as brain neurology. However, we must take care when applying the data from model animals to humans because there are large differences between species, depending on the target field of research.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

This study was designed by K.N., S.T., and Y.S. The data were collected by K.N., R.S., T.O., and T.S.. The manuscript was drafted by K.N. and S.T., and critically reviewed by Y.S., T.O., and H.A.