Journal of Orofacial Sciences

: 2019  |  Volume : 11  |  Issue : 2  |  Page : 79--83

Effects of Vitamin D in Alveolar Bone Remodeling on Osteoblast Numbers and Bone Alkaline Phosphatase Expression in Pregnant Rats During Orthodontic Tooth Movement

Puteri Nazirah B Megat Badarul Hisham, Ida B Narmada, Alida Alida, Dwi Rahmawati, Alexander P Nugraha, Nurul A. R Putranti 
 Orthodontic Department, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

Correspondence Address:
Ida B Narmada
Jl. Mayjen. Prof. Dr. Moestopo 47, Surabaya


Introduction: The vitamin D effect on orthodontic tooth movement in pregnant women remains unknown. The aim of this study was to investigate the post administration of vitamin D effect on osteoblast numbers and bone alkaline phosphatase (BALP) expression in the tension side in pregnant rats during orthodontic tooth movement. Materials and Methods: This study was an in vivo animal experiment; 28 healthy female Wistar rats (Rattus norvegicus) (16–20 weeks’ old) were divided into four groups, with or without intramuscular administration of vitamin D, which were to be observed after 7 and 14 days. Pregnancy was induced with pregnant mare serum gonadotropin and human chorionic gonadotropin. Nickel–titanium coil springs with 30 g/mm2 of force were connected between the right maxillary incisors and the right maxillary first molar. After 7 and 14 days, the animals were sacrificed. Statistical Analysis Used: Analysis of variance with post hoc test (P < 0.05) was performed based on the results of a Levene’s test and a Kolmogorov–Smirnov test (P > 0.05). Results: The highest number of osteoblasts occurred in the C-7 group with mean ± standard deviation of 20.54 ± 8.4. Statistically significant differences were seen in decreased osteoblast number between groups (P = 0.001, P < 0.05). The highest BALP expression was in the E-7 group (3.40 ± 1.625). Nevertheless, no statistically significant difference was observed between the groups (P = 0.240, P > 0.05) in the expression of BALP. Conclusion: The post administration of vitamin D during orthodontic tooth movement in pregnant rats produced no significant enhancement on BALP expression and osteoblast number.

How to cite this article:
Megat Badarul Hisham PB, Narmada IB, Alida A, Rahmawati D, Nugraha AP, Putranti NA. Effects of Vitamin D in Alveolar Bone Remodeling on Osteoblast Numbers and Bone Alkaline Phosphatase Expression in Pregnant Rats During Orthodontic Tooth Movement.J Orofac Sci 2019;11:79-83

How to cite this URL:
Megat Badarul Hisham PB, Narmada IB, Alida A, Rahmawati D, Nugraha AP, Putranti NA. Effects of Vitamin D in Alveolar Bone Remodeling on Osteoblast Numbers and Bone Alkaline Phosphatase Expression in Pregnant Rats During Orthodontic Tooth Movement. J Orofac Sci [serial online] 2019 [cited 2020 Feb 22 ];11:79-83
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Full Text


Orthodontic tooth movement (OTM) is manifested by a biological response to a mechanical stimulus that may subsequently cause changes to bone structure and density through a bone remodeling mechanism.[1] Bone remodeling occurs due to the transition of the microenvironment in the periodontal ligament caused by the secretion of inflammatory mediator cells, growth factors, and neurotransmitters.[2] The bone homeostasis depends on bone formation and resorption by osteoblasts and osteoclasts. Osteoblasts are bone-forming cells involved in bone matrix secretion and synthesis.[3] Bone alkaline phosphatase (BALP) is one of the main enzymes expressed in the early phase of osteoblast maturation and considered a marker of early osteoblast differentiation.[4],[5] The intensification of osteoblastic activity during bone formation is associated with an increase in BALP.[6],[7]

Nowadays, many women were undergoing an orthodontic treatment than men due to aesthetic considerations. Women are associated with female hormones and they have a possibility of pregnancy during orthodontic treatment.[8] Fluctuation in female sex hormones may affect alveolar bone remodeling during OTM. The presence of these sex hormones has a significant effect on oral tissue due to the presence of receptors that can be found in the gingiva, fibroblasts, and osteoblasts in the periodontal ligament.[9],[10] Often pregnant woman are prescribed vitamin supplements, particularly vitamin D, to meet the increased demand that benefits herself and the fetus. Vitamin D can also be consumed by nonpregnant women, and in insufficiency state and deficiency states in both the genders.[11] Vitamin D exerts a stimulatory effect on osteoblasts that helps to stabilize OTM.[12] Previous study reported a 60% increase in OTM after intraligamentary administration of vitamin D3 in experimental subjects.[13] Although there have been several studies on the effects of vitamin D and sex hormones that may enhance bone remodeling, the influence of vitamin D on OTM in pregnant women remains unknown. For this reason, further research is required. Therefore, this study is intended to provide an analysis of the effect of vitamin D on OTM in pregnant rats, the number of osteoblasts, and the expression of BALP.

 Materials and Methods

Ethical approval for this study (Ethical Committee FKG18) was provided by the Ethical Committee Faculty of Dental Medicine of Airlangga University on July 3, 2018. A total of 28 healthy, 16 to 20-week-old female Wistar rats (Rattus norvegicus), with an average weight of 200 to 250 g were used in this study. The research was fully experimental with a posttest only in the control arm. Sample groups were selected by means of simple random number sampling. Each animal was assigned a unique number, which were thoroughly mixed in a hat and picked by blind-folded researchers. The subjects consisted of female Wistar rats (R. norvegicus; n = 7), who were adapted to the environment for 7 days. Sample size (n = 28 for animal models) was based on Lemeshow’s formula to determine minimum sample size. The determination of pregnancy was by means of postmortem clinical examination and abdomen expansion, subsequently confirmed by dissection and observation of the fetus in womb.

Following appropriate statistical calculations, the subjects were divided into four groups along the following lines [Table 1].{Table 1}

These subjects were acclimatized for 7 days in plastic cages with a standard 12-hour light/dark cycle. The temperature and humidity were maintained at 25°C and 50%, respectively. The subjects were fed a diet of finely ground laboratory food ad libitum and had access to drinking water.

The samples were anesthetized by means of an intramuscular injection of a mixture of xylazine hydrochloride (0.03 mL/100 g body weight) and ketamine hydrochloride (0.07 mL/100 g body weight) (Sigma Aldrich, St Louis, MO, USA) prior to the insertion of a spring (American Orthodontic, Sheboygan, WI, USA). Orthodontic force was applied to all groups to induce a mesial movement of the right maxillary first molar. An 8-mm nickel–titanium coil spring (American Orthodontic, Sheboygan, WI, USA) was inserted under general anesthesia between the right maxillary central incisor and the right maxillary first molar. A 0.07 stainless-steel ligature wire (American Orthodontic, Sheboygan, WI, USA) was used to attach the coil spring to the maxillary right first molar and the maxillary right central incisor. The stainless-steel ligature wires were bent around the maxillary central incisor through a hole created in the distocervical side just above the gingival papilla using a round bur. The stainless-steel ligature wires were cemented to the teeth with Glass Ionomer Cement (GC, Japan) until the wires were completely embedded in the bonding material that was then light cured. The nickel–titanium closed coil springs under 30 g/mm2 of force that had been measured by means of a tension gauge were applied to the teeth. The springs were not reactivated during the experiment. The bands were cemented to the teeth with Glass Ionomer Cement (GC, Tokyo, Japan).[8]

After 7 and 14 days of postcoil spring activation, the subjects in each group (experimental and control) were randomly sacrificed by the administering of an overdose of an anesthetic drug. After sacrificing the subjects, their maxillae were dissected and placed in 10% formalin (OneMed, Sidoarjo, Indonesia). After fixation and removal of the springs, the maxilla was decalcified for 21 to 30 days with 10% ethylenediaminetetraacetic acid (OneMed, Sidoarjo, Indonesia) that was replaced every day. The sections were mounted on glass microscope slides and stained with hematoxylin and eosin (Sigma Aldrich, St Louis, MO, USA) to obtain the number of osteoblasts. For each rat, the number of osteoblasts from the five images were first accumulated with the mean of the five sections subsequently being calculated under 400× magnification, with an Olympus Light Microscope (CX23, Olympus, New York, USA) from five different fields of view and examined by, at least, two individuals.

Immunohistochemistry staining was performed on the remaining sections to detect expression levels of BALP. Immunohistochemistry analysis was performed by means of primary antibody (monoclonal antibody anti-alkaline phosphatase, Santa Cruz, Dallas, Texas, USA). Detection was performed using a horseradish peroxidase polymer-based detection system, 3,3′-diaminobenzidine (DAB substrate Kit, Cat No. ab64238, Abcam, Cambridge, MA, USA) brown chromogen, before being counterstained with hematoxylin and eosin. BALP was quantified at 1000× magnification, with a Nikon H600L Microscope (Nikon, Tokyo, Japan) from five different fields of view and examined by, at least, two individuals.

The data were analyzed with Statistical Package for the Social Sciences (SPSS) 22.0 software (IBM Corporation, Chicago, IL, USA). The data were described as means ± standard deviation. The normality of the data was tested using a Kolmogorov–Smirnov test and the homogeneity of variance using the Levene’s test (P > 0.05). One-way analysis of variance continued with Tukey honestly significant different (HSD) was used to compare the number of osteoblasts and BALP between the experimental and the control groups (P < 0.05).


The highest number of osteoblasts was found in the C-7 group [Figure 1] and [Figure 2] whereas BALP expression was found in the E-7 group [Figure 3] and [Figure 4]. Homogeneity and normality test results showed the osteoblast numbers and BALP expression between groups to be homogeneous and normal distributed (P > 0.05). There were significant differences in the osteoblast number between groups (P = 0.001, P < 0.05). There were significant differences in the osteoblast numbers between the C-7 and C-14 (P = 0.000, P < 0.05), C-7 and E-7 (P = 0.005, P < 0.05), and C-7 and E-14 groups (P = 0.005, P < 0.05). There was no statistically significant difference of BALP expression observed between the groups (P = 0.240, P > 0.05). However, no statistically significant difference was detected between the C-14 with E-7 (P = 0.509, P > 0.05), C-14, and E-14 groups (P = 0.473, P > 0.05), and C-14 with E-7 (P = 1.000, P > 0.05).{Figure 1}{Figure 2}{Figure 3}{Figure 4}


The present study revealed that there were significant differences in the osteoblast numbers between the groups. The difference in control measurements between C-7 and C-14 might be explained by the time course of tooth movement that is known to demonstrate the following three stages: the proliferation phase for up to 7 days, maturation for 14 days, and mineralization for 28 days.[14] Osteoblast differentiation may initiate with the triggering of hormones as well as certain growth factors.[15] In the control group, pregnancy hormones such as estrogen produce a significant increase in the average number of osteoblasts on day 7 due to the function of estrogen that affects osteoblast cell proliferation. The average number of osteoblasts decreases significantly on day 14. Subsequent to initiation of the mineralization phase, osteoblasts may further differentiate into osteocytes becoming a bone lining cell or undergo apoptosis.[16] There were significant differences in decreased osteoblast numbers between groups such as the C7 and E-7, C-7 and C-14, and C-7 and E-14 groups. In addition, the administration of vitamin D during pregnancy in the E-14 group showed an insignificant increase in the number of osteoblasts compared to the E-7 group. This effect may be temporal rather than that of vitamin D. This finding is consistent with that of the research conducted by van Driel and van Leeuwen[17] that administration of vitamin D only slightly increases murine osteoblasts through RUNX2 expression that is associated with osteoblast differentiation in murine subjects.

Based on our finding, the highest osteoblast numbers found in C-7 in which no vitamin D was administered was due to the high level of pregnancy hormones such as estrogen and progesterone. Estrogen promotes proliferation of osteoblasts via alpha estrogen receptors that are expressed in osteoblasts. The high level of estrogen and progesterone during pregnancy optimizes proliferation and differentiation of osteoblasts.[18] Vitamin D also promotes an increase in the expression of ectonucleoride pyrophosphate phosphodiesterase and progressive ankylosis (ank) gene osteoblasts, which subsequently causes an increase in pyrophosphate. The latter is an enzyme that inhibits osteoblasts mineralization by reducing bioavailability of phosphate, a substrate for BALP in the maturation phase.[17] In contrast to murine subjects, the administration of vitamin D may affect proliferation, differentiation, and mineralization of osteoblast through the expression of vitamin D receptor in humans. However, this effect may vary depending on various factors such as the duration of treatment and the dose administered.[17]

On the contrary, there was no statistically significant increase in the expression of BALP in the day 14 control group. BALP is an early marker of osteoblast differentiation and an increase in BALP levels may correlate with an increase in bone formation.[5],[19] Increased expression of BALP occurs when osteoblasts differentiate in the early stages of osteogenesis.[20] A slight increase in BALP expression was observed in the E-7 group compared to the C-7 group, owing to the fact that vitamin D inhibits CYP27B1 (25-hydroxyvitamin D2 1-alphanylase) gene and vitamin D receptor expression that promotes BALP differentiation and secretion of preosteoblast.[17] The decreased expression of BALP in the E-14 compared to the E-7 groups may be due to the secretion of BALP into the extracellular matrix when it reached a peak.[5],[19] Several studies imply that BALP expression increases due to osteoblast regulation and subsequently experiences a decrease.[5],[14]

Limitations of the study

The main limitation of this study was the short observational period. The second limitation was the small number of samples. The third limitation was only a few molecular markers that were used in this study. To obtain more accurate findings when comparing the parameters in the study, the variables need to be standardized. Future long-term studies with a higher sample size and a better standardization experimental procedure and more markers for the analysis are recommended.


Our study concluded that the post administration of vitamin D during OTM in pregnant rats produced no significant enhancement on BALP expression and osteoblast number. However, further research is required with more control and better study design to analyze the effect of vitamin D during OTM in pregnant rats to confirm this possibility


The authors would like to thank the Orthodontic Department, Faculty of Dental Medicine and Faculy of Veterinary Medicine, Universitas Airlangga, Surabaya, for the support and help.

Financial support and sponsorship

This study was funded and supported by Penelitian Dosen Pemula (PDP), Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia, with agreement number 86/UN3/2018.

Conflicts of interest

There are no conflicts of interest.


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