Osteocalcin and serum insulin-like growth factor-1 as biochemical skeletal maturity indicators
Tripathi et al. Progress in Orthodontics
Osteocalcin and serum insulin-like growth factor-1 as biochemical skeletal maturity indicators
Tulika Tripathi 0
Prateek Gupta 0
Priyank Rai 0
Jitender Sharma 1
Vinod Kumar Gupta 1
Navneet Singh 0
0 Department of Orthodontics and Dentofacial Orthopaedics, Maulana Azad Institute of Dental Sciences , Bahadur Shah Zafar Marg, New Delhi 110002 , India
1 Department of Biochemistry, Govind Ballabh Pant Institute of
Background: With change in concepts of growth determination methods, there is a surge in the measurement of biomarkers for appraisal of growth status. Osteocalcin is a bone-specific protein and was observed to parallel the normal growth curve. Hence, the present study was intended to assess the levels of serum osteocalcin and serum insulin-like growth factor-1 (IGF-1) and compare them with cervical vertebral maturation index (CVMI) stages. Methods: The cross-sectional study was performed on 150 subjects (75 males and 75 females) in the age group of 8-20 years and segregated into six CVMI stages. Serum osteocalcin and IGF-1 were estimated by ELISA. Mann-Whitney U test was used to compare the mean ranks of serum osteocalcin and serum IGF-1 with different CVMI stages. Spearman correlation was performed to find association between serum osteocalcin and serum IGF-1 across six CVMI stages. Results: Peak serum IGF-1 levels were obtained at CVMI stages 4 and 3 for males and females, respectively, with insignificant difference between stages 3 and 4 in females. Peak serum osteocalcin levels were found at stage 5 and 3 for males and females with insignificant difference from other stages except stages 5 and 6 in males. A statistically significant correlation was seen between serum IGF-1 and serum osteocalcin across six CVMI stages (P < 0.01). Conclusions: Osteocalcin followed IGF-1 across all CVMI stages but showed insignificant interstage differences.
Osteocalcin; IGF-1; Biochemical markers
Successful application of various orthodontic treatment
modalities depends on the skeletal growth status of the
patient. Skeletal growth can be predicted via hand wrist
and cervical vertebral maturation index (CVMI) stages,
but it comes at an expense of radiation exposure to the
patient and inherent limitation of variability in subjective
assessment of the radiographs [
According to functional matrix theory, growth and
development of skeletal units is governed by function of
surrounding soft tissue matrix [
]. Craniocervical relationships
are affected by the associated functions like respiration,
digestion, speech, and equilibrium [
]. Further, it has been
established that cervical column differs in various skeletal
jaw relationships and pressure; morphology of facial
components and body posture alter the height of vertebral
bodies and hence, affect the reliability of CVMI [
With the changing concepts in growth determination
methods, growth can be assessed via measurement of
extracellular bone matrix constituents, which are
molecular signatures of the skeletal growth process [
Insulin-like growth factor-1 (IGF-1) is one such matrix
constituent, which has been profoundly researched for
its role in both prenatal and postnatal skeletal growth. It
facilitates growth under the action of growth hormone
and is also directly stimulated by androgens to accelerate
growth velocity [
]. IGF-1 receptor has even been
localized to mandibular condylar cartilage, thus acting as a
suitable candidate to evaluate the pubertal growth in the
craniofacial region [
]. To consolidate the outcomes of
research on IGF-1, we need to explore new bone-related
factors, which may provide an insight towards precise
skeletal growth assessment during puberty. Among all
bone matrix constituents, only osteocalcin is unique to
bone and can serve as a sensitive and specific serum
marker for bone formation [
Further, bone formation depends on osteoprogenitor
cell replication and differentiation into osteoblasts. Serum
IGF-1 can simultaneously stimulate these two functions
] and has been observed to stimulate osteocalcin
synthesis in bone [
]. IGF-1 affects longitudinal bone growth
while osteocalcin reflects activity of the whole skeleton
and not just the sites of longitudinal growth [
Moreover, osteocalcin is a marker of late osteoblast
] and was found to parallel the growth
velocity curve [
], thus depicting a direct representation of
process of bone formation.
Thus, the aim of the present study was to assess the
levels of serum osteocalcin and serum IGF-1 and to
compare it with CVMI stages. Furthermore, we intended
to determine the significance of the marker levels
between the two genders in different CVMI stages.
This cross-sectional study was conducted on 150 subjects
(75 males and 75 females) in the age group of 8–20 years
who visited the Department of Orthodontics and
Dentofacial Orthopaedics, Maulana Azad Institute of Dental
Sciences, New Delhi, India. Subjects were enrolled in the
study if they were in good general health. Subjects with
growth abnormality, systemic disease, history of long-term
medication, trauma, or surgical intervention in the area of
cervical vertebra were excluded from the study.
Our study was approved by the research ethical
committee of Maulana Azad Institute of Dental Sciences, New
Delhi, India. Written informed consent was obtained from
the subjects and their parents following explanation of the
test procedure orally and through a bilingual patient
The sample size appraisal was done at 5% significance
level (α = 0.05). A minimum of ten subjects were required
in each CVMI stage based on power of 80%. Personal
information, history, and standardized lateral cephalograms
were obtained for all subjects. Two examiners (TT and
PG) independently evaluated the cervical vertebrae
radiographic morphology on lateral cephalograms using the
criteria of Hassel and Farman [
] and grouped the 150
selected subjects into six CVMI stages. A week later,
similar radiographic evaluation was done by the same
examiners for intra-examiner reliability.
Five milliliters of blood was collected from median
cubital vein by venipuncture in red plain blood collection
vials for each subject between 0900 and 1000 hours to
avoid diurnal variations, and the timings were matched
with taking of lateral cephalograms. Assessment of serum
alkaline phosphatase, ionized calcium, and phosphorous
was carried out for all the chosen subjects to further
exclude any skeletal metabolic imbalance. Each blood
sample was centrifuged at 4000 rpm for 15 min to separate
the serum, which was then segregated equally by pipetting
in two different plastic Eppendorf tubes (Eppendorf,
Hamburg, Germany) and stored in different plastic boxes
at −80°C for separate evaluation of IGF-1 and osteocalcin.
Samples were prevented from repeated freeze/thaw cycles.
Serum IGF-1 ELISA (DRG International, USA) and
Microvue Osteocalcin EIA (Quidel Corporation, CA,
USA) kits were used to measure serum IGF-1 and
osteocalcin levels, respectively. Serum samples were subjected
to enzyme-linked immunosorbent assay based on
competitive binding with anti IGF-1 and osteocalcin
antibodies pre-coated on a 96-well plate. Using the mean
absorbance values for each sample, corresponding
concentrations were determined after plotting the standard
calibration curves. The minimum analytical detection
limits for serum IGF-1 and osteocalcin assays were 9.75
and 0.45 ng/ml, respectively.
Statistical analysis was carried out with SPSS software
for Windows (version 23.0; SPSS, Chicago, Ill). The
Kruskal-Wallis and Mann-Whitney U tests were used
to compare mean ranks of serum IGF-1 and osteocalcin
among different CVMI stages. Comparison between
males and females for each CVMI stage was also
performed with the Mann-Whitney U test. The Spearman
correlation test was performed to find relationship
between serum IGF-1 and osteocalcin values at different
CVMI stages. Kappa statistic was used to measure
interexaminer and intra-examiner reliabilities in staging of
Kappa statistics showed no significant difference in
inter-examiner and intra-examiner readings (0.92 and
Mean serum IGF-1 levels in males increased
progressively from lowest value (239.79 ng/ml) at CVMI stage 1
to peak value at stage 4 (528.03 ng/ml) followed by decline
towards stage 6. It was observed that CVMI stages 3 and
5 had comparable means. In females, IGF-1 levels rose
from minimum levels (175.81 ng/ml) at stage 1 to peak
(527.50 ng/ml) at stage 3. The levels declined from stage 3
to stage 6 (Table 1). Peak values for both females and
males were comparably similar while minimum values
were smaller for females as compared to males (Fig. 1).
Table 2 summarizes the mean values and ranges of the
osteocalcin in males and females, respectively. In males,
osteocalcin showed gradual increase from stage 1 to
highest level (6.29 ng/ml) at stage 5 followed by sudden
decline to reach lowest value (2.28 ng/ml) at stage 6. In
females, osteocalcin gradually increased from stage 1 to
reach highest levels at stage 3 (5.26 ng/ml) and declined
thereafter. The peak value for males was found to be
higher than that of the peak value for females (Fig. 2).
The Kruskal-Wallis test showed statistically significant
differences in mean ranks of serum IGF-1 and
osteocalcin levels among CVMI stages. It denoted variable
distributions across the six CVMI stages.
The Mann-Whitney U test performed to reveal
interstage differences in serum IGF-1 values showed that in
males, CVMI stage 1 had statistically significantly lower
value than at stage 3 (P < 0.01), stage 4 (P < 0.001), and
stage 5 (P < 0.01) while CVMI stage 4 IGF-1 value was
significantly higher than all other CVMI stages (P < 0.05).
No significant differences were observed among CVMI
stages 1, 2, and 6 (Table 3).
In females, CVMI stage 1 IGF-1 value was statistically
significantly lower than all other CVMI stages (P < 0.0001).
CVMI stage 2 value was significantly lower than CVMI
stages 3, 4, and 5 (P < 0.0001) but significant difference
was not observed with CVMI stage 6. CVMI stage 3 value
was significantly higher than all CVMI stages (P < 0.0001)
except stage 4 (Table 3).
Inter-CVMI stage difference by performing the
Mann-Whitney U test for osteocalcin showed that
osteocalcin values increased gradually till stage 5 with
insignificant difference between adjacent stages and
declined statistically significantly at stage 6 (P < 0.01) in
males whereas in females, though it was a progressive
rise till stage 3 followed by steady decline till stage 6,
difference was insignificant between adjacent stages
Significant difference between the IGF-1 levels in males
and females was observed at CVMI stages 2 (P < 0.01), 3
(P < 0.01) and 6 (P < 0.0001) while the difference was not
significant in other stages (Table 4). Results of the
MannWhitney U test between males and females revealed a
statistically significant difference in osteocalcin levels at
CVMI stage 5 (P < 0.05) (Table 4).
Spearman correlation coefficient (ρ) was used to
determine the correlation between IGF-1 and osteocalcin
across CVMI stages. A positive correlation was observed
between IGF-1 and osteocalcin during the entire CVMI
staging and was noted to be significant for both males and
females combined at CVMI stages 1 to 6 (P < 0.001), 3 to
6 (P < 0.001), 4 to 6 (P < 0.001), 1 to 5 (P < 0.01), and 5 to
6 (P < 0.01) (Table 5). Further, correlation was statistically
significant between IGF-1 and osteocalcin for all ranges of
CVMI stages (P < 0.05) in males and for CVMI stages 3 to
6 (P < 0.05) and 4 to 6 (P < 0.05) in females (Table 5).
Growth assessment of the craniofacial region is a subject
of interest for the clinicians, researchers, and scientists.
Observational studies involving analysis of series of
cephalometric films and the behaviour of cells and extracellular
matrix molecules are the methods employed in the past
two decades to gain information about growth and growth
]. Radiographic methods though popular, have
limitations in clinical applicability. Thus, the task of
providing information about growth is entrusted to
biomarkers whose levels correlate among different body
fluids at particular growth stage as depicted by Sinha et al.
for IGF-1 [
Until now, IGF-1 has proved to be comparably
authentic in representing growth status as compared to other
biomarkers but disagreement still remains, which initiated
the need for exploring the potentiality of other biomarkers.
Osteocalcin was employed in our study as it is the most
abundant non-collagenous protein of the bone matrix and
is exclusively associated with bone [
In the 1990s, osteocalcin was established as a marker
of bone formation and bone turnover [
with the discovery of osteocalcin’s expression in
differentiated osteoblasts, its role in growth has continuously
Osteocalcin, a vitamin K-dependent protein
undergoes carboxylation to bind hydroxyapatite in bone
and has higher affinity for calcium, thus facilitating
bone mineralization [
]. Thus, circulating osteocalcin
was assayed using two-site ELISA in our study to
assess the growth status during puberty.
Results in our study depicted that IGF-1 and
osteocalcin showed similar changes with skeletal maturation as
pubertal growth curve. Peak values for IGF-1 were
observed at CVMI stage 4 in males and CVMI stage 3 in
females. Many previous studies observed similar results
for both males [
] and females [
Masoud et al. [
] who discovered peak values at CVMI
Peak osteocalcin values were found at CVMI stage 5 in
males and CVMI stage 3 in females. Studies on mice have
provided evidence of the role of osteocalcin in increasing
testosterone production in males by feedforward loop
]. Moreover, higher serum testosterone levels found in
late puberty [
] and significant positive correlation
between osteocalcin and testosterone as reported by
Johansen et al. [
] might be the cause of peak osteocalcin
levels observed at CVMI stage 5 in males in our study.
Earlier peak in females might be linked to rising oestrogen
levels in late puberty as oestrogen causes growth plate
closure and inhibit periosteal apposition [
osteocalcin levels peaked 2 years earlier in females
(13.29 years) as compared to males (15.33 years), which
corresponded to the previous studies [
During puberty, bone grows both longitudinally and in
cortical thickness. Whereas longitudinal bone growth was
reported to cease after mid puberty, cortical thickening
continues in late puberty [
]. Further, osteocalcin has
been observed to be high in cortical bone in comparison
to trabecular bone [
]. Such site specificity with
significant influence of oestrogen is pivotal for observed
sexual dimorphism in skeletal growth. Moreover, higher
peak osteocalcin values observed in males as compared to
females might be associated with greater cortical thickness
observed in males as compared to females . Thus,
different mechanisms with complex interactions between
associated biomarkers may regulate bone growth during
It was reported by Johansen et al. [
] that osteocalcin
correlated significantly with IGF-1. In our study, a
positive correlation was observed between osteocalcin and
IGF-1 across CVMI stages, which was highly significant
in all the stages in males but from CVMI stage 3 to stage
6 in females. Thus, in males, osteocalcin and IGF-1
followed the same trend across all CVMI stages whereas
in females, a similar trend was observed only after peak
levels. Such gender-based correlation involving
particular pubertal stages was not emphasized in previous study
]. Moreover, the increase in osteocalcin levels on
IGF-1 administration indicates a role of IGF-1 on
osteocalcin regulation as reported by Johansson et al. [
Inter-CVMI stage differences revealed that osteocalcin
levels gradually rise in both males and females while
suddenly decline in males from CVMI stage 5 to CVMI
stage 6 which is in contrast to more gradual decline in
females from CVMI stage 3 to CVMI stage 6. Such
gender variation in osteocalcin during puberty suggests
some sex hormone regulation of osteocalcin levels. On
the contrary, IGF-1 showed significant difference in peak
levels at CVMI stages 3 and 4 from other CVMI stages.
Gender differences for osteocalcin at each CVMI stage
were not found to be significant as there was a gradual
rise in both males and females except stage 5 when
males attained peak values and females showed declining
levels. Nevertheless, both males and females exhibited
lowest osteocalcin values at the completion of growth in
CVMI stage 6. Conversely, IGF-1 showed significant
difference between males and females at CVMI stages 3
and 6 with stage 3 values higher in females and stage 6
values higher in males. It demonstrated attainment of
earlier peak in females and prolonged growth spurt in
males. Thus, our results signify that IGF-1 was still
higher in males as compared to females at CVMI stage 6
when osteocalcin levels were lowest and comparable in
both males and females. This might have resulted due to
IGF-1 regulation of both bone and muscle growth [
According to Xu et al. [
], muscle growth peak later than
bone growth, which might be the reason for higher IGF-1
values even in CVMI stage 6 as compared to CVMI stage
1. While at the same time, osteocalcin values declined to
its lowest owing to its bone specificity. Gender differences
in IGF-1 values observed in late CVMI stage could be
explained from findings of Neu et al. [
] who described
gender-specific course in muscle growth during puberty
with males demonstrating prolonged growth as compared
Further, when IGF-1 and osteocalcin were compared,
peak values were observed at different CVMI stages in
males, which pointed towards the role of some other
male specific factor, which is regulating osteocalcin
levels during puberty and may be attributed to the
longer period of cortical growth [
Growth hormone is the common hormone, which
stimulates skeletal growth both via facilitating IGF-1
release and might be involved in production of osteocalcin
in osteoblasts [
]. In the present study, both the
markers indicated skeletal maturity. Although IGF-1
receptors have been identified in mandibular condyle and
local production of IGF-1 proceeds in parallel to
availability to IGF-1 receptors, role of osteocalcin status in
growth regulation of mandibular condyle is still to be
authenticated with evidence [
]. Orthodontics relies
significantly on the status of skeletal growth, which must
be substantiated with backing in the form of multiple
biomarkers. Further research is required to ascertain
whether a particular marker is better than any other
marker to depict growth status or different markers yield
distinct data at a particular growth stage.
1. Serum IGF-1 showed peak levels at CVMI stage 4
and stage 3 in males and females, respectively, with
statistically significant interstage differences except
stages 3 and 4 in females.
2. Serum osteocalcin peak values were observed at
CVMI stage 5 and stage 3 in males and females,
respectively, but with statistically insignificant
interstage differences except stages 5 and 6 in females.
3. A statistically significant correlation was obtained
between serum IGF-1 and osteocalcin across all six
4. A statistically significant difference in serum IGF-1
values was observed between males and females at
CVMI stages 2, 3, and 6 whereas for osteocalcin at
CVMI stage 5.
Contribution of IGF-1 in growth process is important
and could serve as a practically possible marker to assess
growth status. In the light of present findings, role of
osteocalcin in skeletal growth cannot be neglected considering
its bone specificity and its accompanying of growth curve.
Future research involving hormones and bone-related
factors with greater sample size in which subjects should be
longitudinally assessed would uncover the complexity of
pubertal growth regulation and increase our understanding
of facts still inexplicable at present.
The work presented here was carried out in collaboration among all authors.
TT, PR, and VKG defined the research theme and designed the study. TT and
PG conducted the study, performed the literature review, interpreted the
results, and wrote the manuscript. TT and VKG provided guidance in every
phase of study. TT, PR, VKG, and JS guided in the statistical analysis and
interpretation of data. TT, PG, and NS wrote the manuscript and interpreted
the results. TT, PG, JS, PR, VKG, and NS revised the manuscript. All authors
read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Postgraduate Medical Education and Research, Jawaharlal Nehru Marg, New
Delhi 110002, India.
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