Scholarly article on topic 'Effect of dietary calcium deficiency and altered diet hardness on the jawbone growth: A micro-CT and bone histomorphometric study in rats'

Effect of dietary calcium deficiency and altered diet hardness on the jawbone growth: A micro-CT and bone histomorphometric study in rats Academic research paper on "Medical engineering"

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{"Masticatory function" / micro-CT / Histomorphometry / "Midpalatal suture" / "Low-calcium diet"}

Abstract of research paper on Medical engineering, author of scientific article — Yuko Fujita, Shota Goto, Maika Ichikawa, Ayako Hamaguchi, Kenshi Maki

Abstract Objectives We examined the effects of a low-calcium diet and altered diet hardness on bone architecture and metabolism in the maxilla and mandible. Materials and methods Male rats (n =48, 3 weeks old) were divided into six groups. In total, 24 rats were given a normal-calcium diet and the others were given a low-calcium diet. Each group was then divided into three subgroups, which were fed a ‘hard̕ diet for 8 weeks, a ‘soft̕ die for 8 weeks, or switched from the soft diet after 4 weeks to the hard diet for 4 weeks. The bone architecture was analyzed using cephalometry and micro-computed tomography, in addition, the bone metabolism was analyzed using serum bone markers and bone histomorphometry in the maxilla and mandible. Moreover, the bone formation patterns were evaluated using histopathologically in the midpalatal suture. Results The low-calcium diet affected bone architecture by increasing bone turnover and the soft diet affected bone architecture mainly by increasing bone resorption. The soft diet changed the chondrocyte cell layers into fibrous connective tissues in the midpalatal suture. At 4 weeks after the return to a hard diet from a soft diet, recovery of the deterioration in bone architectures was seen in the maxilla and mandible. Conclusions We demonstrated that mastication with a hard diet is effective for recovering the collapsed equilibrium of jaw bone turnover and the deteriorating jaw bone architectures due to the poor masticatory function during the growing period.

Academic research paper on topic "Effect of dietary calcium deficiency and altered diet hardness on the jawbone growth: A micro-CT and bone histomorphometric study in rats"

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Effect of dietary calcium deficiency and altered diet hardness on the jawbone growth: A micro-CT and bone histomorphometric study in rats

Yuko Fujita*, Shota Goto, Maika Ichikawa, Ayako Hamaguchi, Kenshi Maki

Division of Developmental Stomatognathic Function Science, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Japan

ARTICLE INFO

ABSTRACT

Article history:

Received 30 November 2015 Received in revised form 2 May 2016 Accepted 31 August 2016

Keywords:

Masticatory function micro-CT

Histomorphometry Midpalatal suture Low-calcium diet

Objectives: We examined the effects of a low-calcium diet and altered diet hardness on bone architecture and metabolism in the maxilla and mandible.

Materials and methods: Male rats (n = 48, 3 weeks old) were divided into six groups. In total, 24 rats were given a normal-calcium diet and the others were given a low-calcium diet. Each group was then divided into three subgroups, which were fed a 'hard' diet for 8 weeks, a 'soft' die for 8 weeks, or switched from the soft diet after 4 weeks to the hard diet for 4 weeks. The bone architecture was analyzed using cephalometry and micro-computed tomography, in addition, the bone metabolism was analyzed using serum bone markers and bone histomorphometry in the maxilla and mandible. Moreover, the bone formation patterns were evaluated using histopathologically in the midpalatal suture. Results: The low-calcium diet affected bone architecture by increasing bone turnover and the soft diet affected bone architecture mainly by increasing bone resorption. The soft diet changed the chondrocyte cell layers into fibrous connective tissues in the midpalatal suture. At 4 weeks after the return to a hard diet from a soft diet, recovery of the deterioration in bone architectures was seen in the maxilla and mandible.

Conclusions: We demonstrated that mastication with a hard diet is effective for recovering the collapsed equilibrium of jaw bone turnover and the deteriorating jaw bone architectures due to the poor masticatory function during the growing period.

© 2016 Published by Elsevier Ltd.

1. Introduction

Masticatory function is known to influence growth of the craniofacial complex. Many studies have shown that reduced masticatory function resulted in changes with smaller mandibles, with loss of bone mass and thinner condylar cartilage, in growing animals (Hichijo et al., 2014; Kiliaridis, Engstrom, & Thilander, 1985; Mavropoulos, Kiliaridis, Bresin, & Ammann, 2004). In modern societies, the food tastes of pubertal children change worldwide (Kotecha et al., 2013 ; Maki, Nishioka, Morimoto, Naito, & Kimura, 2001; Poti, Duffey, & Popkin, 2014). The increased consumption of softer food, primarily represented by processed foods, leads to a decrease in masticatory force and may cause an increase in malocclusion.

* Corresponding author at: Division of Developmental Stomatognathic Function Science, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manaduru, Kokurakita-ku, Kitakyushu 803-8580, Japan. E-mail address: y-fujita@kyu-dent.ac.jp (Y. Fujita).

http://dx.doi.org/10.1016/j.archoralbio.2016.08.036 0003-9969/® 2016 Published by Elsevier Ltd.

Conventionally, osteoporotic fractures developed with aging. However, in Japan the incidence of bone fractures in primary school children, junior high school students, and high school students increased by approximately three-fold over the past 40 years (Omori, 2010). Three major factors in bone fractures are heredity, lack of exercise, and inappropriate eating habits. In eating habits, inadequate calcium intake can lead to insufficient peak bone mass and increase the risk of bone fractures (Nakagi et al., 2010).

A recent study showed that a 4-week low-calcium diet and a soft diet caused significant deterioration in cortical and trabecular bone structures in both the maxilla and mandible of growing rats (Goto, Fujita, Hotta, Sugiyama, & Maki, 2015). However, the influence of these diets on bone metabolism in the jaw bones during the growing period is not fully understood. Additionally, the effects of a soft diet on bone formation and of altering diet hardness have not been determined. Thus, the aim of this study was to evaluate the effects of dietary calcium deficiency and altered diet hardness on bone structure and bone metabolism in the maxilla and mandible using micro-computed tomography

(micro-CT) and bone histomorphometry in growing rats. Moreover, we evaluated the bone formation patterns histopathological-ly in the midpalatal suture in response to changes in diet hardness.

2. Materials and methods

2.1. Animal care

In total, 48 3-week-old male rats were purchased from Charles River Japan (Kanagawa, Japan). The animals were housed individually under a 12/12-h light-dark cycle, at a constant temperature of 22 ± 1 °C and humidity of 50 ±5%. The rats were divided randomly into six groups (n = 8 each). First, 24 rats were given a normal calcium diet (NCa) containing 510mg/100g calcium (AIN-93G) (Reeves, Nielsen, & Fahey, 1993) while the other rats were given a low-calcium diet (LCa) containing 144 mg/ 100 g calcium (Watanabe, Imamura, Uchikanbori, Fujita, & Maki, 2008). Moreover, they were divided into three subgroups; those given a hard diet for 8 weeks (HD), a soft diet for 8 weeks (SD), or switched from a soft diet after 4 weeks to a hard diet for 4 weeks (SHD). "Hard" diet groups were given the diet in pellet form with a standard hardness (24.1 kg/cm2), while the "soft" diet groups were given powders. All diets were purchased from Oriental Yeast Co., Tokyo, Japan. Rats had ad libitum access to food and tap water throughout the study. Body weight was recorded every week. For dynamic histomorphometry, all rats were given a subcutaneous injection of calcein (8 mg/kg body weight; Kanto Chemical Co., Tokyo, Japan) on days 5 and 1 prior to death. After 8 weeks, all rats were euthanized using pentobarbital sodium, and bilateral femur and craniofacial bones were harvested and cleaned of adherent tissue. All animal procedures were approved by the Committee for Care and Use of Laboratory Animals of Kyushu Dental University (13-20).

2.2. Serum analyses

Blood was obtained from the rats under anesthesia. Serum was isolated by centrifugation (3000 rpm, 15 min) and stored at -80 °C. Serum calcium and phosphorus levels were quantified using routine laboratory methods (Nagahama Life Science Laboratory, Shiga, Japan). Serum levels of osteocalcin, a sensitive biochemical marker of bone formation, were determined using an osteocalcin ELISA kit (Rat Osteocalcin ELISA Kit DS; DS Pharma Biomedical Co., Ltd., Osaka, Japan). Serum levels of C-terminal cross-linked telopeptides of type I collagen (CTX-1), a sensitive marker of bone resorption were quantified using an ELISA kit (RatLaps EIA; Immunodiagnostic Systems, Boldon, UK).

23. Cephalometric analyses

In all rats, growth of the femur and craniofacial bones was evaluated by lateral and dorsoventral cephalometric radiographs using a soft X-ray machine (SOFTEX ESM-2, SOFTEX Co. Ltd., Tokyo, Japan) at 28 kVp, 6 mA, 30-s exposure, and a focus-to-film (New Instant Film, Hanshin Technical Lab., Ltd., Nishinomiya, Japan) distance of 60 cm (Abbassy et al., 2008Abbassy, Watari, & Soma, 2008). A10 mm steel calibration rod was used as an index of length on the film with the femur or the jawbone and was photographed. Using imaging software (ImageJ, available from the NIH website), the total length and the width at the midpoint of the femur were measured in lateral planes. Regarding the jaw bones, the 11 cephalometric landmarks were also digitized (Fig. 1). Selected linear measurements were then obtained Kiliaridis et al. (1985).

To ensure the reproducibility of the measurements, the data were digitized twice at an interval of 2 weeks. Measurement error was calculated according to Dahlberg's double determination method (Dahlberg, 1940). The error variance of linear analyses was less than 0.04 mm.

2.4. Micro-CT

A Scan Xmate-L090 (Comscantecno Co., Kanagawa, Japan) micro-CT instrument was used to prepare representative right maxilla and mandible images in all rats. The measurement sites were selected as a buccal-lingual cross-slice of the first maxillary molar region that connected the centers of the intermediate buccal root and the mesial-palatine root, including the midpalatal suture (Fig. 2a,b). For the mandibles, the measurement sites were selected as a buccal-lingual cross-slice of the first mandibular molar region that connected the centers of the intermediate buccal root and the intermediate lingual root (Fig. 2c,d). Samples were scanned at 10mm resolution, and the total tissue area in the alveolar bone except for the cortical bone region was defined as the total tissue volume (TV; mm2) manually. Threshold values of the trabecular and cortical bones were specified based on the calibration data from measurements of rat bone samples using three-dimensional image analysis TRI/3D-BON software (Ratoc System Engineering, Tokyo, Japan). To distinguish cortical bone and bone marrow in the analysis, cortical bone was specified as areas separated from bone marrow with luminance values of >31 (range, 0-255). Trabecular bone was defined by the bone volume (BV; mm2); such areas were specified by removing dental roots and bone marrow areas from the TV with luminance values in the range of 1-30 (Fig. 2b,d). It is accepred that three-dimensional references included in the TV and BV can be used as primary two-dimensional measurements of area

Fig. 1. Cephalometric landmarks on radiographs.

a, sagittal. Co: the most posterior and superior point on the mandibular condyle. Go: the most posterior point on the mandibular ramus. Mn: the most concave portion of the concavity on the inferior border of the mandibular corpus. Gn: the most inferior point on the ramus that lies on a perpendicular bisector of the line Go-Mn. I1: the most anterior and superior point on the alveolar bone of the mandibular incisor. Mu2: the junction of the alveolar bone and the distal surface of the maxillary third molar. Iu: the most anterior-inferior point on the maxilla posterior to the maxillary incisors.

b, transverse (maxilla). P1 and P2: the most anterior and medial points within the temporal fossae that produce the most narrow palatal width.

c, transverse (mandible). Go1 and Go2: the points on the angle of the mandible that produce the widest width. M1 and M2: the points on the alveolar bone at the mandibular first molar that produce the widest width.

Buccal ^t

Palatal

Buccal

Ml M2 M3

Lingual

\ Lingual

Buccal

Fig. 2. Three-dimensional micro-CT images of the upper and lower first molar regions in rats. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Horizontal view (a) and frontal cross-sectional image (b) of the maxillary first molar region. Horizontal view (c) and frontal cross-sectional image (d) of the mandibular first molar region. White lines indicate the portions of the images used for analysis of the maxilla (a) and mandible (c). Yellow areas indicate cortical bone areas. Blue areas indicate trabecular bone tissue areas (b, d). M1, the first molar, M2, the second molar, M3, the third molar, MB, mesial-buccal root, IB, intermediate buccal root, DB, disto-buccal root, MP, mesial-palatine root, DP, disto-palatine root, IL, intermediate lingual root, M, mesial root, D, distal root.

by the American Society for Bone and Mineral Research (ASBMR) (Bouxsein et al., 2010; Dempster et al., 2013). The trabecular bone volume fraction (BV/TV; %), trabecular thickness (Tb.Th; mm), trabecular number (Tb.N; 1/mm), and trabecular separation (Tb. Sp; mm) were calculated. Regarding cortical bone, the cortical bone area (Ct.Ar; mm2) and marrow area (Ma.Ar; mm2) were measured directly. The total cross-sectional area inside the periosteal envelope (Tt.Ar = Ct.Ar + Ma.Ar; mm2), average cortical thickness (Ct.Th; mm), and cortical bone area fraction (Ct.Ar/Tt.Ar; %) were also calculated (Goto et al., 2015). The error variances of the micro-CT values were less than 0.008 mm2 (TV, BV, Ct.Ar, and Ma.Ar) and 6.5 mm (Ct.Th).

2.5. Specimen preparation

After micro-CT analyses, the trimmed maxillae and mandibles were fixed in 70% ethanol and stained with Villanueva bone stain (Villanueva, 1974). The bone tissues were then dehydrated in ethanol and embedded in methylmethacrylate. Frontal cross sections of the mesial root of first molar region of the maxilla and mandible were cut (300 mm) and polished (30 mm) with a precision bone saw in the same area used in the micro-CT analyses (Exakt cutting grinding system; EXAKT Apparatebau, Norderstedt, Germany). For histomorphometry, one specimen from each rat was prepared for the maxilla and mandible.

After dynamic histomorphometric measurements, each specimen was also stained using the Goldner method (Goldner, 1938) for histopathological evaluation.

2.6. Bone histomorphometry

Images of the maxilla and mandible were scanned under a light/epifluorescence microscope (Carl Zeiss, Thornwood, NY; Fig. 3a-d). Regarding the trabecular and cortical bones, measurement areas are shown in Fig. 3a and b (blue line, periosteal surface of cortical bone; white areas, trabecular bone) (Fujita, Watanabe, Uchikanbori, & Maki, 2011). Histomorphometric parameters were measured at 200 x magnification using a semiautomatic method (HistometryRT Camera; System Supply, Tokyo, Japan; Fig. 3c,d). The measured primary parameters for trabecular bone included bone surface (BS; mm), osteoid surface (OS; mm), osteoblast surface (Ob.S; mm), number of osteoclasts (N.Oc; N), osteoclast surface (Oc.S; mm), single-labeled surface (sLS; mm), and doublelabeled surface (dLS; mm). The static histomorphometric parameters were as follows: OS/BS (%), Ob.S/BS (%), N.Oc/BS (N/100; mm), and Oc.S/BS (%). Ob.S/BS and Oc.S/BS were measured as percentages of the BS. N.Oc/BS was identified and quantitated relative to the BS. The dynamic histomorphometric parameter was determined as follows: mineralizing surface, MS/ BS = (dLS + sLS/2)/BS; %. The measured primary parameter for

Fig. 3. Histological images in the first molar regions of the maxilla and mandible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Frontal cross-section with Villanueva Goldner staining of the maxilla (a) and mandible (b). Blue lines indicate the periosteal surface of cortical bone. The white areas indicate the trabecular bone tissue areas. Measurements for the histomorphometric analysis were made of the interior of the trabecular bone tissue area (trabecular bone) and along the periosteal surface (cortical bone). A higher magnification (200x) of the trabecular bone tissue area in the mandible (c). Arrow heads indicate osteoblasts. Arrows indicate osteoclasts. A fluorescent micrograph with higher magnification (200x) of the periosteal surface in the mandible (d). A green line indicates single-labeled surface. A yellow line (calcein overlaps with the periosteum) indicates double-labeled surface. Frontal cross-section with Villanueva bone staining of the midpalatal suture (e).

cortical bone was induced periosteal bone surface (BS; perimeter), OS, and the lengths of the sLS and dLS. These data were used to calculate OS/BS or MS/BS. The terminology used is in accordance with the ASBMR's Committee on Histomorphometric Nomenclature (Dempster et al., 2013).

Measurement areas of the midpalatal suture are shown in Fig. 3e. The measured parameters for the midpalatal suture areas included the average suture width (mm) and cartilage area (mm2). The average suture width was measured by tracing the interface of the cartilaginous and trabecular bone tissues, corresponding to the strongly stained surfaces with Villanueva bone staining at the bilateral distal edges of the cartilage cell layers. The cartilage area was defined as ossification areas within the cartilage cell layers. We measured the areas where chondrocytes and cartilage matrix were strongly stained with Villanueva bone staining within the cartilage

cell layers (Liu, Tang, Xiao, Liu, & Yu, 2014). The error variances of the all bone histomorphometric parameters were <0.32 mm.

2.7. Statistical analyses

All data are presented as means and standard deviations (SDs). A two-way analysis of variance, followed by a Tukey HSD test, was used to investigate all of the endpoints between dietary calcium intake (LCa and NCa) and between diet hardness (SD, HD, and SHD), as well as any interaction. When significant interactions were found by two-way ANOVA, multiple comparisons with Bonferroni's method were used to explore the exact nature of the interactions among the groups. A result was considered statistically significant at p < 0.05. All statistical comparisons were made using SPSS software (ver. 19.0; IBM Japan, Tokyo, Japan).

Table 1

Body weight, femoral growth and serum parameters.

Body weight

Femoral growth

Serum parameters

Initial (g)

Final (g)

Length (mm) Width (mm)

Calcium (mg/dl)

Phosphorus (mg/dl)

Osteocalcin (ng/ml)

(ng/ml)

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

47.18 ±5.76 47.30 ±4.60 47.58 ±3.93 47.30 ±3.54 47.00 ±4.04 47.57 ± 4.03

429.04 ± 15.76 416.88 ±37.40 423.67 ± 25.24 390.27 ± 15.56 367.76 ± 17.48 383.76 ±31.68

37.53 ± 0.97 37.93 ± 0.59 38.95 ± 1.26 37.43 ± 0.41 37.14 ±0.89 37.60 ± 0.52

4.30 ±0.21 4.52 ±0.34 4.43 ±0.35 3.26 ±0.29 3.13 ±0.32 3.18 ±0.24

11.24 ±0.38 11.36 ±0.76 11.33 ±0.92 10.87 ±0.65 10.23 ±0.67 9.83 ± 0.38

10.38 ± 1.63 10.60 ±2.35 10.30 ±1.12 10.80 ±0.29 10.65 ±0.91 10.50 ±1.23

136.74 ± 10.64 120.92 ±17.75 142.80 ± 12.93 380.00 ±96.87 348.88 ± 95.48 365.45 ±58.60

42.52 ± 10.70 44.63 ± 7.29 56.07 ± 10.85 94.15 ±6.07 79.45 ± 11.47 77.82 ± 12.63

Two way ANOVA with Tukey HSD test (p-value) LCa vs NCa Ns 0.000

SD vs HD Ns Ns

SHD vs HD Ns Ns

Interaction (p-value) Ns Ns

0.000 Ns Ns Ns

0.000 Ns Ns Ns

0.000 Ns Ns Ns

Ns Ns Ns Ns

0.000 Ns Ns Ns

0.000 Ns Ns Ns

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD; soft diet. SHD, switch from soft to hard diets. CTX-1, C-terminal cross-linked telopeptides of type I collagen. Ns, No significance.

Table 2

Cephalometric analysis of the maxilla and mandible.

Parameters (mm)

Maxilla

Mandible

Mu2-Iu

Go1-Go2

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

21.47 i 0.57 21.58 i 0.70 21.79 i 0.51 19.86 i 0.46 20.25 i 0.24 19.52 i 0.60

10.88 i 0.37 10.79 i 0.61 10.56 i 1.15 10.36 i 0.82 10.04 i 0.81 10.71 i 0.74

12.40 i 0.33 11.10 i 0.33 11.80 i 0.51 10.23 i 0.99 8.37 i 0.35 9.72 i 0.34

30.57 i 1.62 28.42 i 1.52 30.62 i 1.18 27.30 i 1.06 24.45 i 1.02 26.29 i 0.27

14.98 i 0.78 14.32 i 0.46 15.02 i 0.56 12.97 i 0.78 10.30 i 0.80" 11.80 i 0.88

20.99 i 1.13 19.62 i 0.26 20.82 i 1.13 18.39 i 1.53 17.23 i 1.15 18.27 i 0.42

9.77 i 0.34 9.07 i 0.19 9.71 i 0.24 8.88 i 0.04 8.13 i 0.30 8.67 i 0.21

Two-way ANOVA with Tukey HSD test (p-value)

LCa vs NCa 0.000 Ns

SD vs HD Ns Ns

SHD vs HD Ns Ns

Interaction (p-value) Ns Ns

0.000 0.000 0.002 Ns

0.000 0.000 Ns Ns

0.000 0.000 Ns

0.000 0.004 Ns Ns

0.000 0.000 Ns Ns

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Ns, No significance. * P<0.05, vs NCa-SD and LCa-HD groups (Multiple comparisons with Bonferroni's method).

3. Results

3.1. Body weight, femoral growth, and serum parameters

At the end of the experiment, the low-calcium diet had affected body weight gain and femoral growth significantly (Table 1). The low-calcium diet resulted in significantly lower levels of serum calcium and significantly higher levels of serum osteocalcin and CTX-1 (Table 1).

3.2. Cephalometric evaluations

The low-calcium diet affected all values measured significantly, with the exception of P1-P2. The SD affected all mandibular parameters significantly, but comparisons between the SHD and the HD groups revealed no statistically significant differences in the values of Co-I1, Co-Gn, Go1-Go2, and M1-M2. A significant interaction and synergistic effect between the LCa and the SD groups was found to affect Co-Gn (Table 2).

3.3. Micro-CT histomorphometry

In the cortical bone of the maxilla, the low-calcium diet and the SD affected the average cortical thickness (Ct.Th) and cortical bone area fraction (Ct.Ar/Tt.Ar) values, whereas comparisons between the SHD and the HD groups revealed no statistically significant difference in those parameters (Table 3; Fig. 4).

Regarding the trabecular bone, the low-calcium diet affected the trabecular bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) values significantly. The SD affected the values of Tb.Th significantly, whereas comparisons between the SHD and the HD groups revealed no statistically significant difference in Tb. Th values. Significant interactions and synergistic effects between the LCa and SD groups were found to affect the values of BV/TV, trabecular number (Tb.N), and trabecular separation (Tb.Sp) (Table 3; Fig. 4).

In the cortical bone of the mandible, the low-calcium diet and the SD affected the values of Ct.Th and Ct.Ar/Tt.Ar significantly. Significant interactions and synergistic effects between the LCa and SD groups were found to affect the values of Ct.Th and Ct.Ar/Tt. Ar (Table 4; Fig. 5).

Regarding trabecular bone, the low-calcium diet affected all trabecular bone parameters significantly. The SD also affected all trabecular bone parameters significantly, whereas comparisons between the SHD and the HD groups revealed no statistically significant difference in the values of BV/TV, Tb.Th, or Tb.N. Significant interactions and synergistic effects between the LCa and the SD groups were found to affect the values of Tb.N and Tb.Sp (Table 4; Fig. 5).

3.4. Bone histomorphometry

In the cortical bone of the maxilla (periosteal bone surface of the palatal side), the low-calcium diet increased the values of OS/

Table 3

micro-CT analysis of cortical and trabecular bones in the maxilla.

Cortical bone

Trabecular bone

Ct.Th (mm)

Ct.Ar/Tt.Ar (%)

BV/TV (%)

Tb.Th (mm)

Tb.N (1/mm)

Tb.Sp (mm)

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

626.49 i 11.94 414.99 i 20.26 531.68 i 39.98 133.91 i 18.98 83.89 i 13.22" 115.36 i 18.60

45.39 i 3.22 38.81 i 2.83 44.04 i 2.28 20.54 i 2.36 14.80 i 2.34 17.60 i 2.54

22.15 i 4.47 23.48 i 3.35 23.31 i 1.75 13.79 i 2.10 7.12 i 0.53" 11.88 i 1.98

177.46 i 8.88 141.25 i 24.14 167.31 i 35.22 69.51 i 5.40 61.65 i 3.77 69.41 i 4.62

1.24 i 0.21 1.68 i 0.20 1.43 i 0.20 1.99 i 0.30 1.16 i 0.08" 1.71 i 0.24

641.66 i 49.10 462.51 i 60.99 547.89 i 87.42 442.99 i 75.61 806.01 i 57.95" 525.30 i 86.05

Two-way ANOVA with Tukey HSD test (p-value)

LCa vs NCa 0.000 0.000

SD vs HD 0.000 0.000

SHD vs HD Ns Ns

Interaction (p-value) 0.000 Ns

0.000 Ns Ns 0.010

0.000 0.031 Ns Ns

0.041 Ns Ns 0.000

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Ct.Th, average cortical thickness. Ct.Ar/Tt.Ar, cortical bone area fraction. BV/TV, trabecular bone volume fraction. Tb.Th, trabecular thickness. Tb.N, trabecular number. Tb.Sp, trabecular separation. Ns, No significance.

* P < 0.05 vs NCa-SD and LCa-HD groups (Multiple comparisons with Bonferroni's method).

1—1—t-(lun) —1—1

Fig. 4. Representative micro-CT images of frontal cross-section of the hard plate in the maxillary first molar region of the rats.

NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD; soft diet. SHD, switch from soft to hard diets. Thinning of cortical bone width and the deterioration of architecture of trabecular bone in the total tissue area were observed in LCa groups. Bone marrow areas in the NCa-SHD group were thinner than those in the NCa-SD group.

BS and decreased the values of MS/BS significantly. The SD increased the values of OS/BS significantly, whereas comparisons between the SHD and the HD groups revealed no statistically significant difference in OS/BS values (Table 5).

Regarding the trabecular bone in the maxilla, the low-calcium diet increased the values of OS/BS, Ob.S/BS, N.Oc/BS, and Oc.S/BS significantly, and decreased the values of MS/BS significantly. The SHD increased the values of N.Oc/BS and Oc.S/BS significantly in rats fed the low-calcium diet. Osteoclasts were not seen in the NCa-HD or -SHD groups. The SHD increased the values of MS/BS significantly (Table 5).

Regarding the cortical bone in the mandible, the low-calcium diet affected the values of MS/BS significantly. The SD and the SHD increased the values of OS/BS significantly.

In the trabecular bone of the mandible, the low-calcium diet increased the values of OS/BS, Ob.S/BS, N.Oc/BS, and Oc.S/BS significantly. The SD increased the values of N.Oc/BS significantly and decreased the values of OS/BS significantly, whereas comparisons between the SHD and the HD groups revealed no statistically significant difference in those parameters (Table 6).

Regarding the midpalatal suture, the low-calcium diet increased the suture width significantly. The SD decreased the suture width significantly, whereas comparisons between the SHD and the HD groups revealed no statistically significant difference in suture width. Cartilage areas were not seen in the SD groups. The SHD increased the cartilage area significantly in rats fed the low-calcium diet (Table 7).

Table 4

Micro-CT analysis of cortical and trabecular bones in the mandible.

Cortical bone Ct.Th (mm)

Ct.Ar/Tt.Ar (%)

Trabecular bone BV/TV (%)

Tb.Th (mm)

Tb.N (1/mm)

Tb.Sp (mm)

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

437.26 ±21.08 318.77 ±17.55 363.81 ±19.85 152.64 ± 10.49 120.61 ± 11.23* 152.34 21.85

36.36 ±1.11 29.93 ± 0.97 32.77 ±1.75 16.12 ±0.92 13.46 ± 1.17* 16.14 1.69

27.19 ±3.99 21.24 ±2.68 24.02 ±4.01 8.82 ± 1.65 3.58 ±0.83 7.67 2.68

151.00 ±15.98 128.59 ±8.71 141.20 ± 19.67 65.27 ±3.25 58.52 ± 2.26 67.97 5.47

1.79 ±0.09 1.65 ±0.13 1.70 ±0.21 1.35 ±0.20 0.61 ±0.15* 1.11 0.30

407.48 ±42.51 480.67 ± 50.02 451.96 ±67.20 607.20 ±32.91 1542.60 ±80.51* 908.06 ±63.92y

Two-way ANOVA with Tukey HSD test (p-value)

LCa vs NCa 0.000 0.000

SD vs HD 0.000 0.000

SHD vs HD 0.000 0.015

Interaction (p-value) 0.000 0.005

0.000 0.001 Ns Ns

0.000 0.021 Ns Ns

0.000 0.000 Ns 0.006

0.000 0.000 0.000 0.000

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Ct.Th, average cortical thickness. Ct.Ar/Tt.Ar, cortical bone area fraction. BV/TV, trabecular bone volume fraction. Tb.Th, trabecular thickness. Tb.N, trabecular number. Tb.Sp, trabecular separation. Ns, No significance.

* P<0.05 vs NCa-SD and LCa-HD groups (Multiple comparisons with Bonferroni's method). y P<0.05 vs NCa-SHD and LCa-HD groups (Multiple comparisons with Bonferroni's method).

mm 1—1—1—1—1 (In) 1

1 ^ 1 NCa-SD J ^^C^HD 1

i—i—i—i— (In) i—i—i—i—i (1mm) H N 1 \ 4 i—i—i—i— (In)

1 LCa-HD 1 Lca-sp M 1 LCa-SHD

Fig. 5. Representative micro-CT images of frontal cross-section of the hard plate in the mandibular first molar region of the rats.

NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. The patterns of bone loss in the LCa and SD groups of the mandible were similar to the same groups in the maxilla. Bone marrow areas in the NCa-SHD group were thinner than those in the NCa-SD group.

3.5. Histopathology

Fig. 6a and b shows histological images of the midpalatal suture. In sections of the palatal suture region in the NCa-HD group, ossification of the cartilage matrix was observed. Cartilaginous areas disappeared and the suture region was filled with fibrous tissue in the sections of the NCa-SD group. However, in the NCa-SHD group, ossification of the cartilage matrix in the midpalatal suture was observed. In sections from the LCa-HD group, the cartilage cell layers were wider than those in the NCa-HD group, although ossification of the cartilage matrix at the bilateral distal edges of the hypertrophic cartilage cell layers was not observed. In sections of the LCa-SD group, chondrocytes decreased in number, and migration of fibroblasts was observed close to the oral cavity in the palatal bone. In sections from the LCa-SHD group, ossification

of the cartilage matrix at the bilateral distal edges of the hypertrophic cartilage cell layers was observed.

4. Discussion

In the present study, we found that the low-calcium diet affected bone architecture by increasing bone turnover, whereas the SD affected bone architecture mainly by increasing bone resorption in the maxilla and mandible of growing rats. In addition, we found that mastication of the HD recovered the collapsed equilibrium of jaw bone turnover due to the SD. Moreover, we found that the SD affected normal chondrocyte development and growth in the midpalatal suture, although suture chondrocytic growth resumed when the diet was switched from soft to hard.

Table 5

Histomorphometric analysis of cortical and trabecular bones in maxilla.

Cortical bone

Trabecular bone

OS/BS (%)

MS/BS (%)

OS/BS (%)

Ob.S/BS (%)

MS/BS (%)

N.Oc/BS (N/100 mm)

Oc.S/BS (%)

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

24.57 ±6.06 67.47 ± 14.61 35.68 ±9.99

53.58 ±17.71 80.10 ±10.52 55.64 ±14.73

41.16 ±4.90 40.86 ±5.68 39.88 ±11.86 34.14 ±10.69 21.94 ±8.97 33.09 ±8.29

10.74 ± 4.03 10.58 ±6.31 17.07 ±8.16 29.41 ± 5.45 26.02 ± 10.37 28.78 ±2.54

12.51 ±9.14 6.02 ± 5.21 0.00 ±0.00 13.64 ± 4.88 13.11 ±8.66 12.24 ±2.07

9.78 ±3.37 11.29± 3.72 13.79 ±0.83 6.42 ±2.32 1.66 ± 1.24 12.23 ±0.66

0.00 ±0.00 44.02 ± 12.76 0.00 ±0.00 10.44 ±23.34 44.54 ±5.39 65.70 ± 36.18*

0.00 ±0.00 0.95 ± 0.56 0.00 ±0.00 0.22 ± 0.49 1.47 ± 1.32 1.74 ±0.77*

Two way ANOVA with Tukey HSD test (p-value)

LCa vs NCa 0.000 0.004

SD vs HD 0.000 Ns

SHD vs HD Ns Ns

Interaction (p-value) Ns Ns

0.000 Ns Ns Ns

0.020 Ns

0.035 Ns

0.004 Ns

0.040 Ns

0.012 0.013 0.020 0.011

0.005 0.008 0.032 0.033

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Ns, No significance.

* P<0.05 vs NCa-SHD and LCa-HD groups (Multiple comparisons with Bonferroni's method).

Table 6

Histomorphometric analysis of cortical and trabecular bones in mandible.

Cortical bone

Trabecular bone

OS/BS (%)

MS/BS (%)

OS/BS (%)

Ob.S/BS (%)

MS/BS (%)

N.Oc/BS (N/100 mm)

Oc.S/BS (%)

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

27.39 ±8.15 55.20 ±10.89 43.29 ±5.69 38.49 ±13.21 53.13 ±11.47 44.20 ±3.00

46.65 ± 5.20 48.39 ±8.49 51.87 ±1.49 42.34 ±2.88 40.83 ± 2.37 45.33 ± 0.73

20.07 ± 5.99 13.50 ±2.71 20.98 ±4.61 34.88 ±3.65 25.56 ±6.64 31.17 ±5.17

4.05 ±2.33 3.13 ±1.01 3.57 ±1.33 14.06 ± 1.78 7.55 ± 2.00 10.85 ±3.34

10.91 ±4.10 12.42 ±2.81 13.07 ±6.72 10.16 ±2.18 8.22 ± 1.77 10.85 ±2.82

27.36 ± 10.93 55.85 ± 34.95 46.39 ± 11.14 53.03 ± 14.20 106.63 ±3.79 58.97 ± 25.37

0.75 ±0.16 1.96 ±0.64 0.98 ±0.38 2.09 ±0.89 2.03 ±0.63 1.59 ±0.39

Two way ANOVA with Tukey HSD test (p-value)

LCa vs NCa Ns 0.012

SD vs HD 0.000 Ns

SHD vs HD 0.042 Ns

Interaction (p-value) Ns Ns

0.000 0.012 Ns Ns

0.000 Ns Ns Ns

Ns Ns Ns Ns

0.011 0.015 Ns Ns

0.042 Ns Ns Ns

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Ns, No significance.

Table 7

Histomorphometric analysis of midpalatal suture.

Suture width (mm)

Cartilage area (mm2)

Groups

NCa-HD

NCa-SD

NCa-SHD

LCa-HD

LCa-SD

LCa-SHD

157.71 ±22.26 146.79 ±22.23 139.11 ±20.50 197.62 ± 12.69 145.54 ±16.58 167.41 ±35.66

Two way ANOVA with Tukey HSD test (p-value) LCa vs NCa 0.013

SD vs HD 0.013

SHD vs HD Ns

Interaction (p-value) Ns

49113.71 ±3541.74 0.00 ±0.00 19683.54 ± 4917.17 500.15 ±303.86 0.00 ± 0.00 30728.17 ± 4701.59"

0.000 0.001 0.000 0.000

Data are mean ± standard deviations. NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Ns, No significance.

* P<0.05, vs NCa-SHD and LCa-HD groups (Multiple comparisons with Bonferroni's method).

According to the present results, a dietary calcium deficiency clearly suppressed body weight gain, skeletal bone growth, and serum calcium levels. It is generally accepted that the serum calcium concentration is controlled within a very narrow range by hormones such as calcitonin and parathyroid hormone, and that it plays important roles in the maintenance of a normal heart beat as well as bone metabolism. A reduced calcium concentration has been implicated in arteriosclerosis and vascular calcification due to impaired systemic mineral metabolism (Lutsey et al., 2014; Weaver, 2006). The present results suggest that parathyroid hormone stimulated the excessive resorption of calcium from bone to compensate for a decreased serum calcium concentration. However, the serum calcium concentration did not reach a normal level. A dietary calcium deficiency also suppressed jaw bone growth, with the exception of the growth of P1-P2, suggesting that lateral growth of the palatal bone was complete by the start of the experiment.

In the cortical bone region, a dietary calcium deficiency resulted in deterioration of the cortical bone structure by inhibiting the formation of a mineralized surface in both the maxilla and mandible. The micro-CT images revealed that a dietary calcium deficiency increased bone resorption on the endosteal bone surface in both the maxilla and mandible.

In the trabecular bone region, a dietary calcium deficiency increased bone resorption significantly, in both the maxilla and mandible. A dietary calcium deficiency increased bone formation

in both the maxilla and mandible. These findings indicate that dietary calcium deficiency induced deterioration of the bone architecture in the maxilla and mandible by increasing bone turnover. These results are consistent with the findings that the serum osteocalcin and CTX-1 levels increased significantly in rats fed the low-calcium diet.

However, a dietary calcium deficiency significantly affected MS/ BS in the maxilla, and tended to affect MS/BS in the mandible, suggesting that increased OS/BS due to a dietary calcium deficiency accumulated with stagnation of mineralization at the trabecular bone surface.

Previous studies indicated little stress transfer to the upper and middle portions of the facial bones during mastication, except for the mandible, in macaques and baboons (Bouvier & Hylander, 1981; Hylander, Picq, & Johnson, 1991). Our study shows that the 8-week SD did not affect the parameters in the maxilla, although it significantly suppressed growth in the posterior direction in the corpus (Go-Mn), in the posterior and superior directions in the condyle (Co-I1), in the superior direction in the ramus (Co-Gn), in the buccal direction in the gonial angle (Go1-Go2), and in the buccal direction in alveolar bone (M1-M2). Goto et al. (2015) reported that growth of the Go-Mn, Co-Gn, and Go1-Go2, which are insertion points of the masseter muscles, was affected in rats fed a 4-week SD. An additional 4-week SD extended the range of inhibition of bone growth from the insertions of the masseter muscles to the condyle and alveolar bone in the mandible. These include the insertions of the lateral pterygoid and buccinator muscles. Thus, we believe that a lack of local mechanical stimulation inhibited the growth of the entire mandible by reducing the quantity and quality of muscles that participate in mastication. However, we found improvement in the growth retardation at these portions, with the exception of Go-Mn, when the rats were switched from a soft to a hard diet.

In cortical bone, in both the maxilla and mandible, the SD accelerated the formation of OS/BS on the periosteal surface. Micro-CT revealed an increase in bone resorption on the endosteal bone surface. These findings indicate significant deterioration in cortical bone structure.

However, a recent study reported that the purpose-made soft pellets used as the SD had little effect on the degree of cortical bone mineralization when compared with the HD (standard pellets) in the mandibles of rabbits (Grunheid, Langenbach, Brugman, Everts, & Zentner, 2011). These findings suggest that differences in the pattern of feeding behavior (e.g. chewing or sucking), rather than differences in diet hardness, are more related to changes in jaw bone structure.

Fig. 6. Representative histological images of the hard plate by Villanueva Goldner staining.

NCa, normal-calcium diet. LCa, low-calcium diet. HD, hard diet. SD, soft diet. SHD, switch from soft to hard diets. Frontal cross-section of the area of midpalatal suture region (a). P, periosteum within the oral region of the midpalatal suture, B, bone, BM, bone marrow, SC, suture cartilages. Scale bar = 200 mm. Higher magnifications of the midpalatal suture (b). Scale bar = 50 mm.

According to the results of Goto et al. (2015), the 4-week SD significantly affected Ct.Th and Ct.Ar/Tt.Ar in the maxilla and mandible; however, 4 weeks after a return to the hard diet from the soft diet, recovery of the reductions in Ct.Ar/Tt.Ar in the maxilla occurred, with improving increases in the OS/BS on the periosteal surface and bone resorption on the endosteal bone surface. In contrast to the maxilla, 4 weeks after the return to the hard diet from the soft diet, reductions in Ct.Th and Ct.Ar/Tt.Ar in the mandible had not recovered in rats fed the normal-calcium diet, suggesting that mastication of the hard diet had more positive effects on the cortical bone in the maxilla than in the mandible.

In the trabecular bone, the SD increased bone resorption and tended to decrease bone formation in the maxilla and mandible. These findings differ from those regarding dietary calcium deficiency.

In addition, the SD affected the trabecular bone architecture in the mandible more than in the maxilla, consistent with the findings of Shimizu et al. (2013). The reason for these findings may be that trabeculae in areas distant from the periodontal ligament were more abundant in the mandible than in the maxilla. Generally, masticatory forces exerted from the teeth are transmitted through the periodontal ligament to the alveolar bone, and trabeculae are arranged in response to stress lines on the bone surface (Proffit, Fields, & Sarver, 2013). In an experimental study, the SD affected the turnover of periodontal ligament collagen and altered bone dynamics of the tooth furcation area in young rats (Jang, Merkle, Fahey, Gansky, & Ho, 2015). Additionally, Lieberman suggested that strains in the mastication of hard food are likely to be high in the alveolar process (i.e., major sites of muscle insertion); such strains then decrease in approximate proportion to the distance from the tooth row (Lieberman, 2011). Consequently, we believe that bone resorption spread mainly along the trabeculae, once the transmission of the mechanical stimulation stopped.

A recent study reported that a 4-week SD resulted in significant deterioration in BV/TV, Tb.Th, and Tb.Sp in both the maxilla and mandible (Goto et al., 2015). However, we observed recovery from the deterioration in trabecular bone volume and structure in the maxilla and mandible at 4 weeks after a return to the hard diet from the soft diet; the increase in bone resorption improved. Moreover, 4 weeks after the return to the hard diet from the soft diet, we observed a significant increase in MS/BS in the maxilla and a tendency for an increase in MS/BS in the mandible, above normal levels. We believe that these processes contributed to the result that the HD rapidly compensated for the loss of bone mass due to the SD. Exceptionally, changing the diet hardness in dietary calcium deficiency resulted in a significant increase in bone resorption in the maxilla. This might have resulted from the rapid increase in MS/BS due to the 4-week HD accompanied by increased bone resorption with high bone turnover rates. Thus, we observed recovery from the deterioration in trabecular bone quality as well as bone quantity in the maxilla and mandible, 4 weeks after a return to the HD from the SD.

In the midpalatal suture, under conditions of dietary calcium deficiency, significant extension of the cartilage cell layers and a low degree of mineralization of the cartilage matrix found in the hypertrophic cartilage cell layers suggest that dietary calcium deficiency caused stagnation in the calcification of the cartilage matrix in the midpalatal suture.

Cephalometric analyses revealed that a dietary calcium deficiency had no effect on palatal width, suggesting that endochondral ossification in the midpalatal suture regions continued after growth in the width of the palate was complete.

Regardless of the calcium content included in the diet, the SD resulted in the disappearance of endochondral ossification areas in the midpalatal suture. Many facial sutures persist throughout life,

with their collagen fibers likely functioning to absorb strains by dissipating tensile and shear forces, especially during mastication, in mammals (Lieberman, 2011). In a clinical study, movement of the tongue contributed to food reduction by applying a shearing force to the food between the tongue and hard palate during processing, and the duration of this with hard food, including peanuts, was significantly longer than that with soft food, including bananas (Hiiemae, 2004). These findings suggest that mechanical stimulation, such as tongue pressure on the palate with a solid diet and the transmission of force from the periodontal support through the hard palate during food processing, are closely related to chondrocytic development and maturity, although factors contributing directly to construction changes within the midpalatal suture due to differences in diet hardness have not been clarified.

Similar to our findings, previous studies have reported that a rapid expansion of orthodontic force increased the migration of cellular fibrous tissue including periosteal cells and blood vessels, to the central portion of the midpalatal suture from oral and nasal cavity sides in small animals (Hou, Fukai, & Olsen, 2007; Ma et al., 2008). However, these expansion forces rapidly replaced cartilage tissues with newly formed bone tissues at the oral and nasal cavity side, which differs from our findings. These findings suggest that in various types of mechanical stimulation, physiological force is most suitable for gradual endochondral ossification in the midpalatal suture. Thus, we believe that chondrocyte development and maturity was reactivated 4 weeks after switching the diet from soft to hard. These observations are consistent with our micro-CT findings that mechanical stimulation by the HD for 4 weeks promoted membranous ossification of the deteriorated bone structure (due to the 4-week SD), in the palatal bone.

5. Conclusions

We demonstrated that mastication of the HD was effective for recovering the collapsed equilibrium of jaw bone turnover and the deteriorating jaw bone architectures due to poor masticatory function during the growing period. We also demonstrated that a lack of mechanical stress affected normal chondrocyte development and growth in the midpalatal suture.

Conflict of interest

The authors declare no conflict of interest. Funding

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 25713064).

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