www.nature.

com/scientificreports

OPEN Associations between physical

physique/fitness in children
and bone development
during puberty: a 4‑year
longitudinal study
Arata Akaike1*, Daisuke Suzuki1, Shinya Okuyama1, Yutaro Kudo1, Hiroyasu Shimizu1,
Sara Takanashi1, Kohei Makino1, Junichi Yokoyama2 & Shigeyuki Nakaji1*

Bone growth is most remarkable during puberty. This study aimed to clarify the effects of physique
and physical strength on bone mineral density and bone metabolism markers during puberty to
help improve bone growth during puberty and prevent future osteoporosis. There were 277 pubertal
participants (125 boys and 152 girls) in this survey from 2009 to 2015, all aged 10/11 and 14/15 years.
The measures included physical fitness/physique indices (such as muscle ratio etc.), grip strength,
bone density (osteo sono-assessment index, OSI), and bone metabolism markers (bone-type alkaline
phosphatase and type I collagen cross-linked N-telopeptide). At 10/11-years-old for girls, a positive
correlation was found between body size/grip strength and OSI. At 14/15-year-old for boys, all body
size factors/grip strength were positively correlated with OSI. The change in body muscle ratio was
positively correlated with change in OSI for both sexes. The height, body muscle ratio and grip
strength at 10/11-year-old were significantly associated with OSI (positively) and bone metabolism
markers (negatively) at 14/15-year-old for both sexes. Adequate physique building after 10/11 years
for boys and before 10/11 years for girls may be effective in increasing peak bone mass.

A healthy life expectancy is the average length of time a person can live in good health with independence in
activities of daily living, as proposed by the World Health Organization (WHO) in ­20001. In Japan, the gap
between healthy life expectancy and average life expectancy is now more than 10 ­years2. Therefore, the "National
Health Promotion Movement in the 21st Century (Healthy Japan 21)" was established to help extend healthy
life ­expectancy3,4. To achieve this aim, it is necessary to delay the time when people will nursing care. The pri-
mary causes of care needs in Japan are locomotive syndrome, frailty, and ­osteoporosis5. Moreover, control of
metabolic syndrome, childhood obesity, frailty, and locomotive syndrome are measures to prevent the need for
nursing ­care6.
It is well-known that habitual moderate exercise is necessary for good health. In order to exercise, the locomo-
tor system, comprising bones, joints, and muscles, must be healthy. Therefore, in 2007, the Japanese Orthopedic
Association defined "locomotive syndrome" as "a condition in which mobility is impaired due to musculoskeletal
disorders and [wherein] the risk of requiring long-term care in the future is high,"7 and has since been working
on preventive measures. However, according to a 2021 white paper aging, fractures and diseases of the musculo-
skeletal ­system8 remain the most common cause of care needs in Japan, accounting for a quarter of all care needs.
In particular, osteoporosis, which causes bone fractures, reportedly affects 7.9% of men and 22.9% of women
over the age of 40 years in ­Japan9,10. Early detection and treatment seem to be the most important ways to
prevent osteoporosis. Bbone mineral density (BMD) assessment is essential for early detection and treatment.
Dual energy X-ray absorption (DXA) has traditionally been used as an indicator for bone evaluation among
various radiological methods. However, since fractures reportedly occur even with high BMD, the 2000 National
Institutes of Health (NIH) Consensus C ­ onference11 recommended adding bone quality as a measure of bone
assessment. However, evaluating bone quality is still difficult.

1
Department of Social Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu‑cho, Hirosaki,
Aomori 036‑8562, Japan. 2Faculty of Sport Science, Nippon Sport Science University, Tokyo 158‑8508,
Japan. *email: [email protected]; [email protected]

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 1

Vol.:(0123456789)
 www.nature.com/scientificreports/

One of the methods to evaluate BMD is ultrasonic measurement (quantitative ultrasound, QUS)12–15. Studies
have also shown that QUS and DXA resuits correlate with each o ­ ther16–27. However, QUS is noninvasive, non-
radioactive, and can be used to evaluate pregnant women and children. Moreover,it has the distinct advantage
over DXA that it is mobile.
Bone is resorbed by osteoclasts and formed by osteoblasts. BMD is maintained if the bone metabolic turnover
is normal and the balance between bone resorption and bone formation is maintained.
In contrast, abnormalities in bone metabolic turnover induce decreased BMD. Therefore, to detect osteopo-
rosis at an early stage, bone metabolism markers are used in Japan to evaluate bone metabolic turnover, bone
metabolism markers are independent BMD-related i­ndicators11 that, include bone formation and resorption
markers. The Fracture Intervention Trial (FIT), which has fracture prevention as an endpoint, reported that
BMD is a bone formation marker rather than a bone resorption m ­ arker16,28. In this study, bone metabolism
markers were also measured to objectively examine the dynamics of bone metabolism. These included bone
formation markers (bone-type alkaline phosphatase, BAP) and bone resorption markers (type I collagen cross-
linked N-telopeptide, NTX).
Adolescence is the peak height velocity age (PHVA), when bones grow rapidly, and BMD reaches its maxi-
mum value (peak bone mass, PBM) at approximately 20 years of a­ go16.
One way to prevent osteoporosis is to have a high PBM. However, because the details of bone metabolism in
adolescents are unknown, specific measures to increase PBM cannot be proposed.
Accordingly, this study aimed to clarify the effects of physique and physical strength on BMD and bone
markers during puberty, when bone growth is most-active.
The features of this study are as follows:

1. This is a 4-year cohort study from the fifth elementary grade to the third-year of junior high school.
2. Bone metabolism markers were measured in third-year junior high school students.
3. Body composition was measured and examined separately using the body fat ratioand body muscle ratio.

Methods
Participants. Participants included teens boys and girls who took the Iwaki Health Promotion Project Ele-
mentary and Junior High School Health Survey in both fifth grade in elementary school and third year of junior
high school.
Participants were selected as follows:

1. Four elementary and junior high schools in the Iwaki district of Hirosaki City, located in the north of Japan,
were chosen. The survey was conducted in autumn.
2. From 2009 to 2011, surveys and measurements were conducted on fifth graders (10/11-year-old students)
who provided their consent, as did of their parents. Of the 395 subjects, 361 participated in the survey, with
a participation rate of 91.4%.
3. From 2013 to 2015, surveys and measurements were conducted on third-year junior high school students
(14/15-year-old students) who provided their consent, as did their parents. Of the 415 subjects, 380 partici-
pated in the survey, with a participation rate of 84.3%.
4. A total of 323 students who fulfilled (2) and (3) were selected.
5. Of the 323 participants, those with a history of cardiovascular disease, diabetes, dyslipidemia, or hyperten-
sion, those on medication, those with a history of bone fractures, those with a history of calcaneal fractures,
and those with missing values in the analysis items were excluded. A total of 277 pubescent teens (125 boys
and 152 girls) were included in the analysis.

The components of the survey were a questionnaire, bone density measurement, blood test (bone metabolism
marker), and physical fitness measurement. The survey was conducted within one day in elementary school and
1–2 days in junior high school. The survey took a total of 5 days.

Measures. Questionnaire. A self-administered questionnaire was provided in advance. Participants were

asked to complete it out with their parents or guardians, and the questionnaire was collected on the day of the
measurement. Four public health experts reviewed the responses, and if they had any questions, they checked
with the child or his/her parents. The questionnaire items included age, gender, medical history, current medical
history, and medication status.

Anthropometric evaluation (height and body composition measurements). Height and body composition meas-
urements were taken on the day of the survey as part of the physique assessment.
The body composition measurements included body weight, body fat ratio (% fat), and body muscle ratio (%
muscle). A body composition analyzer based on the bioelectrical impedance method (TBF-110; Tanita Corpora-
tion, Tokyo) was used for measurement. This device uses multiple frequencies of 5 kHz, 50 kHz, 250 kHz, and
500 kHz and has been used in many studies on ­adults29–31. The instrument was designed to measure participants
who are at least 110 cm tall and at least 6 years of age.

Bone ­evaluation32,33. BMD is a major component of bone toughness. BMD evaluation was performed by QUS
using an ultrasonic bone evaluation device (AOS-100NW; Aloka Co., Ltd., Tokyo, Japan). The measurement
site was the calcaneus bone, which was evaluated through the acoustic bone assessment value (Osteo Sono-

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 2

Vol:.(1234567890)
www.nature.com/scientificreports/

Assessment Index, OSI). The speed of sound (SOS) and transmission index (TI) were measured by this device
­ ensity34,35, whereas TI was used
and then used to calculate OSI. SOS was used to measure calcification and bone d
12,15
to measure broadband ultrasound attenuation, a bone quality assessment ­index . OSI was calculated using the
formula:

OSI = TI × SOS2 ,
thus reflecting the characteristics of both SOS and TI. Hence, OSI is considered one of the global index values
in acoustic bone a­ ssessment36.

Muscle strength evaluation. To evaluate muscle strength, we used grip strength, which is ­considered37,38 to
reflect whole-body muscle strength. We followed the method of the "New Physical Fitness Test"39 by the Sports
Agency of the Ministry of Education, Culture, Sports, Science and Technology.
The Smedley type grip strength meter(T. K. K. 5401; Takei Scientific Instruments Co., Ltd., Niigata, Japan).
was used to measure grip strength, with grip width adjusted such that the proximal interphalangeal joint of the
second finger was flexed at 90°. For measurement, the limb position was a standing position with both legs open,
the pointer of the grip strength meter was held so that it faced outward, and the shoulders were slightly laterally
displaced to not touch the body. Subsequently, the participant was asked to grasp the grip strength meter with
full force while exhaling. During measurement, the participants were asked to hold the grip strength meter still
while maintaining the basic posture. Measurement was performed twice on each hand and, were taken alternately
on the left and right hands, with the best value being adopted.

Measurement of bone metabolism markers. Blood samples were taken from children in their third year of junior
high school in the early morning on an empty stomach, and blood analysis was outsourced to LSI Medience Co.,
Ltd. The company also conducted the measurements for bone formation (BAP) and bone resorption markers
(NTX) using the CLEIA (Chemiluminescent enzyme immunoassay) method.

Statistical analyses. All analyses were performed according to sex.

The measures obtained from the fifth grade of elementary school and those from the third-year of junior high
school were compared using paired t-tests.
To examine potential confounding factors, the correlations between OSI and height, % fat, % muscle, and
grip strength were confirmed for each grade using a partial correlation coefficient. For participants in their
third year of junior high school, the correlation between OSI, BAP, and NTX was confirmed using the partial
correlation coefficient.
To investigate the effect of changes in physique and physical strength on OSI from the fifth grade of elemen-
tary school to the third year of junior high school, the changes in % fat, % muscle, and grip strength in relation
to the change in OSI were examined using a multiple regression analysis. For this analysis, the change in OSI was
used as an objective variable, and the change in each item was used as an explanatory variable.
Odds ratios with 95% confidence interval were calculated using logistic regression analysis to assess the
relationship between physique and physical parameters during the fifth grade of elementary school and bone
metabolism (OSI, BAP, and NTX) during the third year of junior high school.
Height, % fat, % muscle, and grip strength were used as physique/physical fitness indicators for students in
fifth-grade elementary school, with each indicator used to divid students into tertiles of low-value, medium-
value, and high-value groups.
SPSS 16.0J software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses, and a p-value < 0.05 was
considered statistically significant.

Ethical considerations. This study was conducted according to the principles of the Declaration of Hel-
sinki.
The purpose of the study, the right to withdraw from the study at any point, and the data management
methods (including data privacy protection and data anonymity) were thoroughly explained to all participants,
and written consents were obtained from either the participants themselves or from their parents/caregivers.
The Iwaki Health Promotion Project Elementary and Junior High School Health Survey was approved by the
Institutional Review Board of the Graduate School of Medicine, Hirosaki University (approval numbers 2009-
048, 2010-084, 2011-111, 2013-339, 2014-060, and 2015-075).
This study was registered in the University Hospital Medical Information Network (UMIN-CTR, https://​
www.​umin.​ac.​jp; examination name: Iwaki Health Promotion Project Medical Examination; and UMIN Exam
ID: UMIN000040459).

Results
Physical fitness and physique indices, bone mineral density, and bone metabolism markers
(Tables 1 and 2). Among boys, all items except % fat significantly increased, whereas among girls, all items
significantly increased. Bone metabolism marker values in the third year of junior high school were also signifi-
cantly higher in boys than in girls, indicating that bone metabolism during this period was more active in boys
than in girls.

Correlation in each grade (Tables 3 and 4). For girls in the fifth grade, a positive correlation was found
between body size/grip strength and OSI. However, this trend was not observed among boys.

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 3

Vol.:(0123456789)
 www.nature.com/scientificreports/

Characteristics 10/11-year-olds 14/15-year-olds p-value

Height (cm) 142.5 ± 6.7 167.8 ± 6.8 0.000
Body weight (kg) 37.0 ± 9.4 57.7 ± 10.9 0.000
Body fat ratio (%) 17.2 ± 10.8 17.9 ± 7.7 0.160
Body muscle ratio (%) 28.3 ± 3.6 44.2 ± 4.8 0.000
Grip strenght (kg) 17.8 ± 3.4 32.1 ± 5.4 0.000
OSI (× ­105) 2.6 ± 0.4 2.9 ± 0.4 0.000
BAP (µg/L) – 57.6 ± 38.7 –
NTX (nmol BCE/mmol) – 77.7 ± 31.5 –

Table 1.  Baseline characteristics of participants (boys, N = 125). Paired-samples t-test. SD, standard deviation;
p, probability value; N, number of participants; OSI, osteo-sono assessment index; BAP, bone specific alkaline
phosphatase; NTX, type I collagen cross-linked N-telopeptide.

Characteristics 10/11-year-olds 14/15-year-olds p-value

Height (cm) 144.5 ± 6.7 157.2 ± 5.7 0.000
Body weight (kg) 37.4 ± 7.4 51.7 ± 8.3 0.000
Body fat ratio (%) 20.0 ± 7.1 30.2 ± 6.7 0.000
Body muscle ratio (%) 27.9 ± 3.5 33.6 ± 3.2 0.000
Grip strength (kg) 17.6 ± 3.3 22.6 ± 3.9 0.000
OSI (× ­105) 2.5 ± 0.2 3.0 ± 0.4 0.000
BAP (µg/L) – 23.0 ± 15.5 –
NTX (nmol BCE/mmol) – 35.4 ± 13.8 –

Table 2.  Baseline characteristics of participants (girls, N = 152). Paired-samples t-test. SD, standard deviation;
p, probability value; N, number of participants; OSI, Osteo-sono assessment index; BAP, bone specific alkaline
phosphatase; NTX, type I collagen cross-linked N-telopeptide.

Height % fat % muscle Grip strength OSI BAP NTX

r p R p r p r p r p r p r p
Height 0.287 0.001 0.886 0.000 0.451 0.000 0.075 0.204
% fat 0.287 0.001 0.485 0.000 0.348 0.000 0.058 0.259
10/11-year-olds % muscle 0.886 0.000 0.485 0.000 0.594 0.000 0.080 0.186
Grip strength 0.451 0.000 0.348 0.000 0.594 0.000 0.060 0.254
OSI 0.075 0.204 0.058 0.259 0.080 0.186 0.060 0.254
Height 0.060 0.252 0.732 0.000 0.363 0.000 0.148 0.049 − 0.445 0.000 − 0.406 0.000
% fat 0.060 0.252 0.460 0.000 0.256 0.002 0.274 0.001 − 0.025 0.390 − 0.209 0.010
% muscle 0.732 0.000 0.460 0.000 0.651 0.000 0.429 0.000 − 0.122 0.088 − 0.339 0.000
14/15-year-olds Grip strength 0.363 0.000 0.256 0.002 0.651 0.000 0.390 0.000 − 0.142 0.057 − 0.454 0.000
OSI 0.148 0.049 0.274 0.001 0.429 0.000 0.390 0.000 − 0.328 0.000 − 0.331 0.000
BAP − 0.025 0.390 − 0.122 0.088 − 0.142 0.057 − 0.328 0.000 − 0.445 0.000 0.366 0.000
NTX − 0.209 0.010 − 0.339 0.000 − 0.454 0.000 − 0.331 0.000 − 0.406 0.000 0.366 0.000

Table 3.  Correlation between each parameter (boys, N = 125). Partial correlation coefficient. The 10/11-year-
olds were fifth-grade elementary school students, whereas the 14/15-year-olds were third-year junior high
school students. N, Number of participants; r, partial correlation coefficient; p, probability value; % fat, body fat
ratio; % muscle, body muscle ratio; OSI, osteo-sono assessment index; BAP, bone specific alkaline phosphatase;
NTX, type I collagen cross-linked N-telopeptide.

For third-year junior high school boys, all body size factors/grip strength were positively correlated with OSI
and negatively correlated with NTX and /BAP. In contrast, this trend was less pronounced among girls.

Relationship between changes in physical fitness and physique indices and changes in BMD
over 4 years (Tables 5 and 6). The change in body muscle ratio was positively correlated with the change
in OSI among both sexes.

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 4

Vol:.(1234567890)
www.nature.com/scientificreports/

Height % fat % muscle Grip strength OSI BAP NTX

r p R p r p r p r p r p r p
Height 0.207 0.005 0.885 0.000 0.659 0.000 0.405 0.000
% fat 0.207 0.005 0.463 0.000 0.130 0.055 0.188 0.010
10/11-year-olds % muscle 0.885 0.000 0.463 0.000 0.672 0.000 0.525 0.000
Grip strength 0.659 0.000 0.130 0.055 0.672 0.000 0.356 0.000
OSI 0.405 0.000 0.188 0.010 0.525 0.000 0.356 0.000
Height − 0.095 0.242 0.781 0.000 0.532 0.000 0.154 0.058 − 0.075 0.359 0.093 0.255
% fat − 0.095 0.242 0.210 0.009 0.071 0.385 0.157 0.054 − 0.178 0.028 − 0.202 0.013
% muscle 0.781 0.000 0.210 0.009 0.619 0.000 0.374 0.000 − 0.097 0.236 − 0.094 0.250
14/15-year-olds Grip strength 0.532 0.000 0.071 0.385 0.619 0.000 0.242 0.003 − 0.137 0.093 − 0.061 0.455
OSI 0.154 0.058 0.157 0.054 0.374 0.000 0.242 0.003 − 0.067 0.411 − 0.189 0.020
BAP − 0.075 0.359 − 0.178 0.028 − 0.097 0.236 − 0.137 0.093 − 0.067 0.411 0.376 0.000
NTX 0.093 0.255 − 0.202 0.013 − 0.094 0.250 − 0.061 0.455 − 0.189 0.020 0.376 0.000

Table 4.  Correlation between each parameter (girls, N = 152). Partial correlation coefficient. The 10/11-year-
olds were fifth-grade elementary school students, whereas the 14/15-year-olds were third-year junior high
school students. N, Number of participants; r, partial correlation coefficient; p, probability value; % fat,
body fat ratio; % muscle, body muscle ratio; OSI, Osteo-sono assessment index; BAP, bone specific alkaline
phosphatase; NTX, type I collagen cross-linked N-telopeptide.

Objective variable Explanatory variable B Standard error β p Adjusted ­R2

Change in height − 0.065 0.013 − 0.618 0.000 0.354
Change in % fat 0.003 0.008 0.036 0.750
OSI
Change in % muscle 0.058 0.018 0.408 0.001
Change in grip strength 0.009 0.009 0.086 0.321

Table 5.  Relationship between changes in physical fitness/physique indices and changes in BMD over 4 years
(boys, N = 125). Multiple regression analysis. Adjusted by values at 10/11-year-olds (fifth-grade elementary
school students). B, partial regression coefficient; β, standard partial regression coefficient; p, probability value;
BMD, bone mineral density; N, Number of participants; OSI, osteo-sono assessment index; % fat, body fat
ratio; % muscle, body muscle ratio.

Objective variable Explanatory variable B Standard error β p Adjusted ­R2

Change in height − 0.033 0.013 − 0.458 0.013 0.172
Change in % fat 0.002 0.007 0.022 0.804
OSI
Change in % muscle 0.069 0.023 0.510 0.003
Change in grip strength 0.011 0.0011 0.099 0.303

Table 6.  Relationship between changes in physical fitness/physique indices and changes in bone mineral
density over 4 years (girls, N = 152). Multiple regression analysis. Adjusted by values at 10/11-year-olds (fifth-
grade elementary school students). B, partial regression coefficient; β, standard partial regression coefficient; p,
probability value; BMD, bone mineral density; N, Number of participants; OSI, osteo-sono assessment index;
% fat, body fat ratio; % muscle, body muscle ratio.

Relationship between physical fitness and physique index at 10/11 years of age and bone
metabolism at 14/15 years of age (Tables 7 and 8). There was a tendency for significantly higher
odds ratios in the maximum height, % fat, % muscle,and grip strength groups for higher OSI in third year of
junior high school at third-year junior high school among fifth-grade students.
Furthermore, higher height, %fat, %muscle, and grip strength in fifth grade for both males and females tended
to have significantly lower odds ratios for the BAP and NTX results in ninth grade.

Discussion
Bone repeated formation and resorption occur throughout life. These bone metabolic activities are regulated
by various ­hormones40–46 and cytokines. Bone growth has two peaks: the primary growth phase until the age
of 5 years and the secondary growth phase during the teenage years. During the secondary growth phase, the
bone finishes growing in the long-axis direction, the epiphyseal line closes, and the trabecular bone becomes

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 5

Vol.:(0123456789)
 www.nature.com/scientificreports/

OSI BAP NTX

Tertile value (n) OR 95% CI p OR 95% CI p OR 95% CI p
126.7–139.5 (41) – – – – – – – – –
Height (cm) 139.6–145.0 (42) 1.841 [0.500–6.771] 0.359 1.004 [0.993–1.015] 0.517 0.993 [0.979–1.007] 0.302
145.1–158.5 (42) 7.057 [1.952–25.506] 0.003 0.989 [0.978–1.001] 0.078 0.963 [0.945–0.981] 0.000
3.0–10.3 (41) – – – – – – – – –
% fat (%) 10.5–18.8 (41) 1.612 [0.485–5.356] 0.172 0.993 [0.982–1.004] 0.229 1.000 [0.987–1.014] 0.998
19.0–57.0 (41) 3.387 [3.387–10.872] 0.040 0.984 [0.972–0.996] 0.008 0.965 [0.947–0.984] 0.000
10/11-year-olds
20.85–26.90 (32) – – – – – – – – –
% muscle (%) 26.95–29.45 (44) 6.095 [1.412–26.310] 0.015 0.997 [0.986–1.008] 0.586 0.984 [0.969–0.999] 0.033
29.50–40.90 (49) 24.581 [5.350–112.929] 0.000 0.980 [0.967–0.992] 0.002 0.945 [0.924–0.967] 0.000
9.4–15.9 (32) – – – – – – – – –
Grip strength (kg) 16.0–18.0 (44) 1.570 [0.356–6.923] 0.551 0.981 [0.967–0.995] 0.008 0.993 [0.979–1.007] 0.309
19.0–28.0 (49) 10.764 [2.505–46.260] 0.001 0.969 [0.955–0.984] 0.000 0.977 [0.962–0.993] 0.004

Table 7.  Odds ratios of physical fitness/physique index at 10/11 years of age for OSI/ bone metabolism
at 14/15 years of age (boys, N = 125). Logistic regression analysis. The 10/11-year-olds were fifth-grade
elementary school students, whereas the 14/15-year-olds were third-year junior high school students. OR, odds
ratio; p, probability value; % fat, body fat ratio; % muscle, body muscle ratio; OSI, osteo-sono assessment index;
BAP, bone specific alkaline phosphatase; NTX, type I collagen cross-linked N-telopeptide.

OSI BAP NTX

Tertile value (n) OR 95% CI p OR 95% CI p OR 95% CI p
127.9–141.9 (54) – – – – – – – – –
Height (cm) 142.1–147.4 (49) 10.349 [2.761–38.792] 0.001 0.973 [0.947–1.000] 0.047 0.962 [0.933–0.992] 0.014
147.5–163.3 (49) 9.789 [2.616–36.629] 0.001 0.944 [0.913–0.975] 0.001 0.945 [0.912–0.979] 0.002
8.6–16.5 (51) – – – – – – – – –
% fat (%) 16.6–20.5 (50) 0.904 [0.312–2.623] 0.853 0.974 [0.949–1.001] 0.056 0.965 [0.936–0.995] 0.021
20.6–47.5 (51) 1.099 [0.387–3.120] 0.859 0.953 [0.924–0.983] 0.002 0.961 [0.931–0.991] 0.013
10/11-year-olds
18.55–26.20 (50) – – – – – – – – –
% muscle (%) 26.30–29.35 (48) 8.010 [1.980–32.410] 0.004 0.956 [0.926–0.986] 0.004 0.944 [0.912–0.978] 0.001
29.50–39.10 (54) 19.066 [4.666–77.911] 0.000 0.948 [0.918–0.979] 0.001 0.934 [0.901–0.969] 0.000
9.8–15.8 (51) – – – – – – – – –
Grip strength (kg) 16.0–19.0 (53) 3.542 [1.079–11.628] 0.037 0.987 [0.963–1.011] 0.286 0.971 [0.943–0.999] 0.040
19.3–25.0 (48) 5.541 [1.652–18.592] 0.006 0.952 [0.922–0.983] 0.003 0.931 [0.895–0.969] 0.000

Table 8.  Odds ratios of physical fitness / physique index at 10/11 years of age for OSI/ bone metabolism at
14/15 years of age (girls, N = 152). Logistic regression analysis. The 10/11-year-olds were fifth-grade elementary
school students, whereas the 14/15-year-olds were third-year junior high school students. OR, odds ratio; p,
probability value; % fat, body fat ratio; % muscle, body muscle ratio; OSI, osteo-sono assessment index; BAP,
bone specific alkaline phosphatase; NTX, type I collagen cross-linked N-telopeptide.

denser, which improves BMD. The participants in this study were in the period of secondary sexual charac-
teristics development, when the secretion of sex hormones is active and factors that affect bone metabolism
are entangled. Rauchenzauner et al.47 reported that bone metabolism during puberty exhibits a large variation
with according to age and sex, and that both BAP and tartrate-resistant phosphatase, a bone resorption marker,
decrease after the age of 15 years. However, a study focusing on these factors in Japanese adolescents has not
yet been conducted. Reports of trends in bone metabolism markers and factors associated with DXA among
Japanese adolescents are also very l­imited48. One reason for this is the unwillingness of parents and guardians
to allow invasive tests such as blood sampling and exposure to radiation on their children in conditions that do
not involve diagnosis or treatment.
The main results of this study are:

1. For girls in fifth grade, a positive correlation was found between body size/grip strength and OSI. However,
this trend was not observed among boys. This suggests that the development of body size during early ado-
lescence influences OSI among girls.
2. For third year junior high school boys, all body size factors/grip strength were positively correlated with OSI.
In contrast, this trend was less pronounced among girls, with only change in % muscle and grip strength

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 6

Vol:.(1234567890)
www.nature.com/scientificreports/

positively correlating with OSI. Change in body muscle ratio was positively correlated with the change in
OSI among both sexes. These results suggest that for boys, growth in body size/muscle power from the fifth
grade of elementary school to the third year of junior high school influences OSI.
3. The height, body muscle ratio and grip strength in fifth-grade elementary school were significantly positively
associated with OSI and significantly negatively associated with bone metabolism markers in the third year
of junior high school for both sexes. These findings suggest that the development of body size (height and
body muscle ratio) and grip strength in early adolescence influences OSI and bone metabolism.

The peak height velocity age (PHVA) during the second growth period among Japanese people is observed at
13 years old for boys and 11 years old for girls, with the rate of growth in height being higher in b­ oys49. At age 17
for boys and 15 for girls, the closure of the epiphyseal line begins and BMD increases toward PBM. Considering
the results of the present study in light of this background, we suggest that increasing height, muscle mass, and
muscle strength by fifth grade for girls and after fifth grade for boys, is important for increasing PBM.
Previous studies in growing children and adolescents have reported that both bone resorption and bone
formation markers eventually become ­elevated50. This may reflect active bone metabolism.
The relationship between bone metabolism and BMD has been the subject of many studies among a­ dults51,52.
Although there are some ­reports53–56 with slightly different trends in men, an overview of the results of previous
studies can be summarized as follows: “Bone metabolism markers increase during growth and subsequently,
decrease until the 40 s, remaining unchanged until old age”.
In Japan, the reference values for BAP are 3.7–20.9 µg/L for healthy men and 2.9–14.5 µg/L for healthy pre-
menopausal women. The reference values for NTX are 9.5–17.7 nmol BCE/L for healthy men and 7.5–16.5 nmol
BCE/L for healthy premenopausal ­women57. Compared to these reference values, both markers were elevated
in our study among thirdyear junior high school students and this was more pronounced in boys. This indicates
that bone metabolism is active in thirdyear students, especially in boys. The reason for the gender difference
could be that the third-year boys are still in the growth phase and the epiphyseal line has not yet closed, whereas
in girls, the epiphyseal line is nearer to closure during this period. That is, boys in the third year of junior high
school are still growing and bone growth is active, whereas girls are at the end of their bone growth period and
reaching bone maturity. The trends of bone metabolism markers obtained in this study are consistent with peak
height velocity age in the Japanese p ­ opulation16.
Furthermore, the results of this study suggested that those with large and strong physique and physical
strength in the fifth grade of elementary school had the peak of bone metabolism at a younger age.
However, one limitation of the present study is that the effect of menstruation was not considered. Since bone
metabolism is affected by sex hormones, it is desirable to examine the effect of menstruation in future research.
Based on the findings above and in light of peak height velocity age, we believe that it is important to practice
a lifestyle that will increase sufficient height and muscle mass by the third year of junior high school for males and
by the fifth grade for females to increase peak bone mass. This requires a regular lifestyle, balanced nutritional
intake, and appropriate exercise. We believe that such a lifestyle will lead to the control of childhood obesity,
which is one of the factors leading to the need for nursing care. Moreover, developing desirable lifestyle habits
in childhood may also encourage people to be healthy. Raising awareness of health promotion in childhood and
practicing desirable lifestyle habits may also lead to the suppression of metabolic syndromes, frailty, and locomo-
tive syndromes, which have been identified as a measure for preventing long-term care. To extend healthy life
expectancy, it is important to work on these issues from early adolescence.

Conclusions
Adolescents aged 10/11 years to 14/15 years in the Iwaki area of the Aomori Prefecture were evaluated and fol-
lowed up for four years. Bone metabolism marker values among participants aged 14/15 years were significantly
higher in boys than in girls. At 10/11 years of age for girls, a positive correlation was found between body size/
grip strength and OSI. At 14/15 years of age for boys, all body size factors/grip strength were positively correlated
with OSI. Change in body muscle ratio was positively correlated with the change in OSI among for both sexes.
The height, body muscle ratio and grip strength at 10/11 years of age were significantly positively associated
with OSI and significantly negatively associated with bone metabolism markers at 14/15 years of age for both
sexes. In conclusion, adequate physique building after 10/11 years of age for boys and before 10/11 years of age
for girls may be effective in increasing PBM.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author, S.N., upon
reasonable request.

Received: 28 February 2022; Accepted: 28 July 2022

References
1. WHO. Healthy life expectancy. https://p ​ latfo
​ rm.w
​ ho.i​ nt/d
​ ata/m
​ atern
​ al-n
​ ewbor​ n-c​ hild-a​ doles​ cent-a​ geing/a​ geing-d
​ ata/a​ geing-h
​ ealt​
hy-​life-​expec​tancy.
2. WHO. Life expectancy and Healthy life expectancy Data by country. https://​apps.​who.​int/​gho/​data/​view.​main.​SDG20​16LEXv?​
lang=​en.
3. Ministry of Health. Labour and Welfare. A basic direction for comprehensive implementation of National. Health Promot. https://​
www.​pbhea​lth.​med.​tohoku.​ac.​jp/​japan​21/​pdf/​kenko​unipp​on21_​01e.​pdf.

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 7

Vol.:(0123456789)
 www.nature.com/scientificreports/

4. Tsuji, I. Epidemiologic research on healthy life expectancy and proposal for its extension: A rev. English version of Japanese in the
Journal of the Japan Medical Association. JMA. J. 148, 1781–1784 (2019).
5. Ministry of Health, Labour and Welfare. Comprehensive survey of living Conditions. https://​www.​mhlw.​go.​jp/​engli​sh/​datab​ase/​
db-​hss/​cslc-​index.​html (2019).
6. National Institute of Health and Nutrition. Health Japan 21 (the second term). https://​www.n ​ ibioh
​ n.g​ o.j​ p/e​ iken/k​ enkou
​ nippo
​ n21/​
en/ (2021).
7. Nakamura, K. A “super-aged” society and the “locomotive syndrome”. J. Orthop. Sci. 13, 1–2 (2008).
8. Cabinet Office & Government of Japan. Education, culture, sports and life. https://​www8.​cao.​go.​jp/​kourei/​white​paper/w-​2021/​
zenbun/​03pdf_​index.​html (2021).
9. Yoshimura, N. et al. Cohort profile: Research on osteoarthritis/Osteoporosis Against Disability study. Int. J. Epidemiol. 39, 988–995
(2010).
10. Yoshimura, N. et al. Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: The
research on osteoarthritis/osteoporosis against disability study. J. Bone Miner. Metab. 27, 620–628 (2009).
11. NIH Consensus Development Panel on Osteoporosis Prevention. Diagnosis, and therapy. JAMA 285, 785–795 (2001).
12. Glüer, C. C., Wu, C. Y., Jergas, M., Goldstein, S. A. & Genant, H. K. Three quantitative ultrasound parameters reflect bone structure.
Calcif. Tissue Int. 55, 46–52 (1994).
13. Glüer, C. C. et al. Site-matched calcaneal measurements of broad-band ultrasound attenuation and single x-ray absorptiometry:
Do they measure different skeletal properties?. J. Bone Miner. Res. 7, 1071–1079 (1992).
14. Black, D. M. et al. Axial and appendicular bone density predict fractures in older women. J. Bone Miner. Res. 7, 633–638 (1992).
15. Langton, C. M., Palmer, S. B. & Porter, R. W. The measurement of broadband ultrasonic attenuation in cancellous bone. Eng. Med.
13, 89–91 (1984).
16. Takase, H. et al. Relationship between visceral fat accumulation and metabolic components by sex and age in workplace setting:
A Japanese study of visceral adiposity and lifestyle information-utilization and evaluation. J. Jpn. Soc. Study Obes. 26, 245–253
(2020) (in Japanese).
17. Collinge, C. A., Lebus, G., Gardner, M. J. & Gehrig, L. A comparison of quantitative ultrasound of the calcaneus with dual-energy
x-ray absorptiometry in hospitalized orthopaedic trauma patients. J. Orthop. Trauma 24, 176–180 (2010).
18. Harada, S., Hayashi, T., Otsuka, K. & Nishimura, S. Comparison of the results of screening for osteoporosis using various measuring
methods of bone mineral density. Comparison between lumbar DXA, forearm DXA, calcaneus SXA, and calcaneus USD. Orthop.
Traumatol. 47, 1288–1290 (1998).
19. Duquette, J., Honeyman, T., Hoffman, A., Ahmadi, S. & Baran, D. Effect of bovine bone constituents on broadband ultrasound
attenuation measurements. Bone 21, 289–294 (1997).
20. Han, S. et al. Ultrasound velocity and broadband attenuation as predictors of load-bearing capacities of human calcanei. Calcif.
Tissue Int. 60, 21–25 (1997).
21. Langton, C. M., Njeh, C. F., Hodgskinson, R. & Currey, J. D. Prediction of mechanical properties of the human calcaneus by
broadband ultrasonic attenuation. Bone 18, 495–503 (1996).
22. Han, S., Rho, J., Medige, J. & Ziv, I. Ultrasound velocity and broadband attenuation over a wide range of bone mineral density.
Osteoporos. Int. 6, 291–296 (1996).
23. Serpe, L. & Rho, J. Y. The nonlinear transition period of broadband ultrasound attenuation as bone density varies. J. Biomech. 29,
963–966 (1996).
24. Nicholson, P. H. F., Haddaway, M. J. & Davie, M. W. J. The dependence of ultrasonic properties on orientation in human vertebral
bone. Phys. Med. Biol. 39, 1013–1024 (1994).
25. Massie, A., Reid, D. M. & Porter, R. W. Screening for osteoporosis: Comparison between dual energy x-ray absorptiometry and
broadband ultrasound attenuation in 1000 perimenopausal women. Osteoporos. Int. 3, 107–110 (1993).
26. Palacios, S., Menéndez, C., Calderón, J. & Rubio, S. Spine and femur density and broadband ultrasound attenuation of the calcaneus
in normal Spanish women. Calcif. Tissue Int. 52, 99–102 (1993).
27. Lees, B. & Stevenson, J. C. Preliminary evaluation of a new ultrasound bone densitometer. Calcif. Tissue Int. 53, 149–152 (1993).
28. Black, D. M. et al. Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Fracture
Intervention Trial Research Group. Lancet 348, 1535–1541 (1996).
29. Dimitri, P., Wales, J. K. & Bishop, N. Fat and bone in children: differential effects of obesity on bone size and mass according to
fracture history. J. Bone Miner. Res. 25, 527–536 (2010).
30. Bauer, D. C. et al. Change in bone turnover and hip, non-spine, and vertebral fracture in alendronate-treated women: The fracture
intervention trial. J. Bone Miner. Res. 19, 1250–1258 (2004).
31. Pietrobelli, A. et al. Appendicular skeletal muscle mass: Prediction from multiple frequency segmental bioimpedance analysis.
Eur. J. Clin. Nutr. 52, 507–511 (1998).
32. Morita, R. et al. Quantitative ultrasound for the assessment of bone status. Osteoporos. Int. 7, S128–S134 (1997).
33. Hans, D., Njeh, C. F., Genant, H. K. & Meunier, P. J. Quantitative ultrasound in bone status assessment. Rev. Rhum. Engl. Ed. 65,
489–498 (1998).
34. De Terlizzi, F. D., Battista, S., Cavani, F., Canè, V. & Cadossi, R. Influence of bone tissue density and elasticity on ultrasound
propagation: An in vitro study. J. Bone Miner. Res. 15, 2458–2466 (2000).
35. Hans, D. et al. Ultrasound velocity of trabecular cubes reflects mainly bone density and elasticity. Calcif. Tissue Int. 64, 18–23
(1999).
36. Tsuda-Futami, E. et al. An evaluation of a new gel-coupled ultrasound device for the quantitative assessment of bone. Br. J. Radiol.
72, 691–700 (1999).
37. Ikeda, N. et al. The relationship between grip strength and physical function in elderly community-dwelling women. Rigakuryoho
Kagaku. 26, 255–258 (2011) (in Japanese).
38. Wind, A. E., Takken, T., Helders, P. J. M. & Engelbert, R. H. H. Is grip strength a predictor for total muscle strength in healthy
children, adolescents, and young adult?. Eur. J. Pediatr. 169, 281–287 (2010).
39. Ministry of Education, Culture, Sports, Science and Technology. The new physical fitness test. https://​www.​mext.​go.​jp/a_​menu/​
sports/​stami​na/​03040​901.​htm (1999).
40. Wang, Q. J. et al. Influence of physical activity and maturation status on bone mass and geometry in early pubertal girls. Scand. J.
Med. Sci. Sports. 15, 100–106 (2005).
41. Watanabe, Y. et al. Intravenous pamidronate prevents femoral bone loss and renal stone formation during 90-day bed rest. J. Bone
Miner. Res. 19, 1771–1778 (2004).
42. Fuchs, R. K., Bauer, J. J. & Snow, C. M. Jumping improves hip and lumbar spine bone mass in prepubescent children: A randomized
controlled trial. J. Bone Miner. Res. 16, 148–156 (2001).
43. Chandani, A. K. et al. Bone mineral density and serum levels of aminoterminal propeptide and cross-linked N-telopeptides of
type I collagen in elderly men. Bone 26, 513–518 (2000).
44. Bilezikian, J. P., Morishima, A., Bell, J. & Grumbach, M. M. Increased bone mass as a result of estrogen therapy in a man with
aromatase deficiency. N. Engl. J. Med. 339, 599–603 (1998).
45. Kraemer, W. J. et al. Hormonal and growth factor responses to heavy resistance exercise protocols. J. Appl. Physiol. 69, 1442–1450
(1990).

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 8

Vol:.(1234567890)
www.nature.com/scientificreports/

46. Sutton, J. R., Coleman, M. J., Casey, J. & Lazarus, L. Androgen responses during physical exercise. Br. Med. J. 1, 520–522 (1973).
47. Rauchenzauner, M. et al. Sex-and age-specific reference curves for serum markers of bone turnover in healthy children from 2
months to 18 years. J. Clin. Endocrinol. Metab. 92, 443–449 (2007).
48. Scopacasa, F. et al. Bone density and bone-related biochemical variables in normal men: A longitudinal study. J. Gerontol. A Biol.
Sci. Med. Sci. 57, 385–391 (2002).
49. Ministry of Education. School health statistics. https://​www.e-​stat.​go.​jp/​dbview?​sid=​00031​46920 (2016).
50. Fukunaga, M. Bone metabolism markers. Med. Rev. 114 (2003) (in Japanese).
51. Miller, P. D., Hochberg, M. C., Wehren, L. E., Ross, P. D. & Wasnich, R. D. How useful are measures of BMD and bone turnover?.
Curr. Med. Res. Opin. 21, 545–554 (2005).
52. Garnero, P., Sornay-Rendu, E., Claustrat, B. & Delmas, P. D. Biochemical markers of bone turnover, endogenous hormones and
the risk of fractures in postmenopausal women: The OFELY study. J. Bone Miner. Res. 15, 1526–1536 (2000).
53. Meier, C., Liu, P. Y., Handelsman, D. J. & Seibel, M. J. Endocrine regulation of bone turnover in men. Clin. Endocrinol. 63, 603–616
(2005).
54. Meier, C., Nguyen, T. V., Center, J. R., Seibel, M. J. & Eisman, J. A. Bone resorption and osteoporotic fractures in elderly men: The
dubbo osteoporosis epidemiology study. J. Bone Miner. Res. 20, 579–587 (2005).
55. Szulc, P., Garnero, P., Munoz, F., Marchand, F. & Delmas, P. D. Cross-sectional evaluation of bone metabolism in men. J. Bone
Miner. Res. 16, 1642–1650 (2001).
56. Szulc, P. & Delmas, P. D. Biochemical markers of bone turnover in men. Calcif. Tissue Int. 69, 229–234 (2001).
57. Nakaji, S. et al. Report of the Iwaki Health Promotion Project (2020) (in Japanese)

Acknowledgements
We would like to express our sincere gratitude to all participants, parents, and elementary and junior high school
staffs in the Iwaki area who understood the purpose of this research and kindly cooperated in the preparation
of this paper. This work was supported by the Centre of Innovation Program from the Japan Science and Tech-
nology Agency (grant number: JPMJCE1302). We would like to thank Editage (http://​www.​edita​ge.​com) for
English language editing.

Author contributions
A.A. designed the study, analyzed and interpreted the data. D.S., S.O., Y.K., H.S., S.T., K.M., and J.Y. contributed
to data acquisition. S.N. contributed to the data interpretation. All authors reviewed and edited the manuscript
and approved the final version.

Competing interests
This research was carried out with the support of the Center of Innovation Program (JPMJCE1302) of the Japan
Science and Technology Agency (JST), Japan.

Additional information
Correspondence and requests for materials should be addressed to A.A. or S.N.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

© The Author(s) 2022

Scientific Reports | (2022) 12:13427 | https://doi.org/10.1038/s41598-022-17623-z 9

Vol.:(0123456789)