This cross-sectional study evaluated 52 orthodontic patients aged between 10 and 14 years presenting to the Orthodontic Department at Shahid Beheshti School of Dentistry, following the appropriate institutional ethics approval (IR.SBMU.RIDS.REC.1394.11). The study design was in accordance with the Helsinki Declaration of Human Rights. All the patients had panoramic radiographs and lateral cephalograms obtained for their orthodontic treatment, therefore eliminating the need for unnecessary radiation exposure during the research. All the patients and their parents were informed about the study and the parents signed the informed consent forms.
The sample size was calculated to be 51 (N=17 in each group) using Power and Sample Size Calculation software version 3.0.43. The inclusion criteria consisted of an age between 10 and 14 years, having panoramic radiographs and lateral cephalograms taken for orthodontic treatment, willingness to participate in the study and presence of signed informed consent forms by the parents.
The exclusion criteria were sunbathing more than once a week, taking vitamin D supplements, consumption of vitamin D-rich foods and drinks more often than once a day since the initiation of orthodontic treatment, a history of systemic diseases, medication intake, craniofacial syndromes, bilateral missing of mandibular teeth, poor quality of radiographs, and not performing blood test in the designated laboratory. It should be mentioned that the exclusion criteria were evaluated according to the parents’ responses to the questions.
After obtaining the parents’ consent, all patients underwent blood tests in the designated laboratory to determine their serum 25-hydroxy vitamin D level. All blood tests were performed during six months in fall and winter to eliminate the effect of seasonal changes in vitamin D levels. The patients and their parents were informed of the test results and were educated about the role of vitamin D in the body. The test results were provided to the parents, and they were referred for medical consultation if needed. The patients were classified into three groups according to their 25-hydroxy vitamin D serum level:
Normal: Vitamin D > 30 ng/mL; sufficient vitamin D
Low: 15 < Vitamin D ≤30 ng/mL; moderate vitamin D deficiency
Very low: Vitamin D ≤ 15 ng/mL; severe vitamin D deficiency
To determine the skeletal age of patients, CVM stage analysis of the second, third and fourth cervical vertebrae (CV2-CV4) on lateral cephalograms (Soredex® Cranex-D, Tuusula, Finland) was performed according to Baccetti’s modified protocol . The radiographs were converted to JPEG file format using Digora® software version 8.2 (Strasbourg, France) and observed using Photoshop software (Adobe®, San Jose, CA, USA). Magnification of ×150 was used for more accurate observation when required. In order to prevent errors in the determination of skeletal age, the evaluation of all lateral cephalograms was confined to the cervical vertebrae by the observer to determine the skeletal age without observing the teeth. Two calibrated orthodontists, blinded to the chronological age and serum vitamin D level of patients, independently determined the CVM stage and the skeletal age. In cases of disagreement, a third blind observer also reviewed the radiographs. For each patient, two variables, including the presence/absence of concavity in the inferior border of CV2, CV3, and CV4; and the body shape of CV3 and CV4 (trapezoidal, horizontal rectangular, square, and vertical rectangular) were evaluated, as presented by Baccetti . According to these two variables, the patients were assigned to one of the skeletal maturation stages (CVM1 to CVM6) as presented in Figure 1.
The panoramic radiographs (Soredex® Cranex-D, Tuusula, Finland) were also independently evaluated by two calibrated orthodontists, blinded to the chronological age and serum vitamin D level of patients, to determine the dental age according to Demirjian’s method . For this purpose, the calcification stage of seven permanent teeth in the mandibular left quadrant (from tooth number 24 to 19) was scored individually from A to H. The standard scores of teeth were separately reported for males and females. A total score of dental maturation, which was the sum of individual scores, was reported for each individual. This total score was converted to dental age (in years) according to a standard table. The scores given to different calcification stages of teeth were determined according to Demirjian and Goldstein, as presented in Figure 2 .
Data were analyzed using SPSS software version 21.0 (SPSS® Inc., IL, USA). After the primary assessment of all radiographs, 10 radiographs were randomly selected and re-evaluated after two weeks to assess the intra-observer error. The kappa coefficient was also calculated to assess the inter-observer agreement. The mean kappa coefficient for the intra-observer agreement was also calculated for dental age and skeletal age determination.
The mean dental age of patients was compared among the groups using a t-test. The linear regression model was applied to compare dental age among the three groups after controlling for age and gender. Since the CVM stage was a descriptive variable, an ordinal logistic regression model was used to compare the skeletal age among the groups after controlling the chronological age and gender. The Pearson’s correlation test was used to assess the correlation between dental age and chronological age in each group. P<0.05 was considered statistically significant.
 Joint Photographic Experts Group
 Statistical Package for the Social Sciences