Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion

ABSTRACT Objective: This study aimed to evaluate the effects of systemic teriparatide on sutural bone formation after premaxillary suture expansion in rats. Material and Methods: Twenty Wistar male rats (8-10 weeks old) were randomly divided into two groups, namely, control (C, n=10) and teriparatide (T, n=10). An expansion force was applied to the maxillary incisors using helical spring for a seven-day expansion period, for both groups. On the eighth day, the rats were kept for a seven-day consolidation period, and then 60 µg/kg teriparatide (once a day) was administered to group T subcutaneously for seven days. Then, all the rats were sacrificed, and histological sections were stained with hemotoxylin-eosin for examination. Anti-osteonectin, anti-osteocalcin, anti-Vascular endothelial growth factor (VEGF) and anti-transforming growth factor beta (TGF-β) were evaluated by immunohistochemical analysis in the midpalatal suture area. Results: Histologically, the newly formed bone tissue was observed to be larger in group T than in group C. The number of immunoreactive osteoblasts for osteonectin, osteocalcin and VEGF antibodies was significantly higher in group T than in group C (p = 0.0001). The TGF-β antibody showed a mild reaction in group T, but did not reach significance in comparison with group C (p ˃ 0.05). Conclusion: Systemic teriparatide application following the premaxillary expansion of the suture area may stimulate bone formation and add to the consolidation of the expansion in rats by regulating osteonectin, osteocalcin and VEGF.


INTRODUCTION
Expanding the midpalatal suture using rapid maxillary expansion (RME) appliances is commonly utilized in orthodontic treatment of malocclusions exhibiting a narrow maxillary jaw and posterior crossbite. The main principle of RME appliances is to rapidly expand the upper dental arch, followed by the active bone formation in the midpalatal suture. [1][2][3] After RME, the stabilization of the expansion obtained is required because the upper jaw tends to revert to its previous form, despite a consolidation period. 3 This relapse rate after RME has been reported in the literature to be as high as 63%. 4 Many reasons have been reported to cause this relapse in the maxilla. The main reason has been suggested to be the tension in the midpalatal area and in the surrounding sutures. Nevertheless, the bone organization in the midpalatal suture completed in 3-12 months was reported to prevent this relapse. 5 The amount of bone formed in the midpalatal suture could also shorten the period of consolidation, 3 as previously shown in the literature. 6 Teriparatide is a synthetic derivative of the parathyroid hormone Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion 5 previously used to slow down osteoporosis-related bone loss and to treat fractures without cortical and trabecular bridges.
It can increase osteoblastic activity by modulating the nuclear kappa B ligand/osteoprotegerin (RANKL/OPG) and the insulin-like growth factor, previously found to be involved in bone metabolism. [8][9][10] Teriparatide was also found to promote bone formation during orthodontic treatments for prosthetic purposes 11 and periodontitis-related bone loss. 12 Biochemical markers are indicative of complex bone remodelling processes. Osteocalcin is a pro-osteoblastic non-collagen protein found in bone and dentin, and it plays a role in bone building and metabolic regulation. 13 Its expression is also induced by teriparatide. [14][15][16] Osteonectin, a non-structural matricellular glycoprotein secreted by osteoblasts, binds calcium in bone 17 and may also be increased by teriparatide. 18,19 VEGF 20 and TGF-β 13 are involved in tissue regeneration and reproductive functions activated by teriparatide. 21 Several experimental studies evaluated the effects of different agents, such as resveratrol, propolis and lithium, on bone formation in expanded sutures. [22][23][24] However, according to our knowledge, no study has evaluated the efficiency of teriparatide on bone formation in experimentally expanded sutures. Given the beneficial effects of teriparatide on osteoblastic activity and the origin of teriparatide as a synthetic derivative of Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion 6 a hormone produced by the human body, this study aimed to evaluate the effects of systemic teriparatide application on the bone formation of premaxillary suture in rats treated with maxillary expansion.

ANIMALS AND STUDY GROUPS
Twenty 8-10-week-old male Wistar rats weighing 200±15 g were randomly and equally divided into two groups: group T (n=10), which was treated with teriparatide following the placement of the expansion appliance; and group C (n=10), which served as the healthy control group with only the expansion appliance and without teriparatide administration. During the experimental period, all the rats were kept in separate cages in a quiet room with a controlled temperature (23°C) and a 12 h light and dark cycle. The rats were fed a standard and solid diet, and had access to tap water ad libitum. After appliance placement, they were fed with a softened rat diet, in order not to use their teeth and break the appliance. The Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion 7 The sample size for each group was calculated on the basis of an alpha significance level of 0.05 to achieve 90% power to detect significant differences with a 0.40 effect size (G*Power version 3.0.10; Franz Faul Universidad, Kiel, Germany). 23 The power analysis showed that 20 samples in the parallel group were required, and animals were randomly divided into two groups.

APPLIANCE PLACEMENT
The rats were checked by a veterinarian, who confirmed that their general health conditions were normal. The animals were weighed for weight change before and after the experiment.
The upper incisors of the rat were preferred because of their ease of application and their usability to simulate the maxillary expansion in previous studies. 1,5,6,23 In the maxilla of each rat, the distance between the mesial edges of the two incisors of the rats was measured in the cervical level twice with a caliper (Fig 1), after the expansion (T1) and after the consolidation (T2), initially assumed to be 0 mm (T0).
Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion Figure 1: Measured values between the mesial edges of the two incisors of the subject after the expansion period (E) and the consolidation period (C). The Mann-Whitney U and Friedman tests were used for statistical analysis. No difference was found for the distance (p ˃ 0.05). T-E = Teriparatide group-Expansion period; T-C = Teriparatide group-Consolidation period; C-E = Control group-Expansion period; C-C = Control group-Consolidation period).
The animals were anesthetized with a combination of xylasine  (Figs 2A and 2B). A composite adhesive (Transbond XT, Seefeld, Germany) was used circularly to the helicals, to increase the retention of the springs (Fig 2C).
The springs were placed on a grid and activated on a single arm with a plier. Then they were adjusted with a force gauge (Dentaurum, Ispringen Germany) to give 1.176 N 25 when placed on the mesial of the incisors, and were not reactivated during the seven-day expansion period. 3 After the expansion period, the experiment was conducted with a seven-day consolidation period. At the end of the expansion period, the springs were removed and controlled to be passive and then replaced as a retainer during the retention ( Fig 2D).
After the consolidation, the distance between the mesial edges of the upper incisors was measured with a manual caliper (Dentaurum, Ispringen, Germany). 22 This distance was also measured after the expansion period.
Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion

TERIPARATIDE APPLICATION
Teriparatide is only available in one form (Forsteo ® , Eli and Lilly Company, Indiana, USA) in the market. According to the manufacturer's protocol, during the seven-day consolidation period, 60 μg/kg daily teriparatide 26,27 was injected subcutaneously into the animals of group T. The animals in group C were injected subcutaneously physiological saline (60 μg/kg, 0.9% NaCl) during the consolidation period.

HISTOLOGIC ANALYSIS
All the rats were sacrificed with 200 mg per kilogram of sodium pentobarbital (Pentothal, Abbot, North Chicago, Ill). The premaxillae were resected as a block and fixed in 10% formalin.
Then, the retaining wires were removed, and the premaxillae were decalcified with tetraacetic acid for three weeks. After decalcification, the premaxillae were cut into parts containing two incisor teeth. These incisors served as the primary guide for orienting the sections. Two points were chosen after cutting the section to the sagittal plane perpendicularly. The first one was located at the alveolar crest, and the second one was located 4 mm apical to the crest. This imaginary line passed through the center of the incisor crown at its gingival portion. 22 The paraffin blocks were sectioned serially at 5-μm intervals,

RESULTS
At the beginning of the study, 24 Wistar rats were included, but three rats were lost after general anaesthesia. The remaining 21 rats were observed to tolerate the expansion well during expansion and consolidation. No abnormal findings, in terms of general health conditions, were found in the weight measurements made at the beginning and at the end of the experiment. On the sixth day of the consolidation phase, one eye infection was observed in a rat, which was removed from the study. Finally, the study was completed with 20 rats (10 rats in each group).
No statistically significant difference was found in the comparison of body weights within and between groups before the experiment and before being sacrificed (p > 0.05) (Fig 3). No significant difference was observed within and between groups for the distance between the mesial edges of the teeth at the end of the expansion protocol and before sacrifice (p > 0.05) (Fig 1).
Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL FINDINGS
All sections stained with H&E were examined in the upper jaw suture region under a light microscope. The new bone formation was observed in group T (Fig 4) at a more intense level than that in group C (Fig 5) in visual evaluation. In the suture of group C, new bone areas were observed in the form of islands and extensions. Conversely, in group T, bone formation was shown as merged wide areas.    7). No statistically significant difference was found between the groups for TGF-β antibody osteoblastic immunoreactivity (p ˃ 0.05 ; Fig 7), but a slight antibody staining was observed in group T that did not reach significance (Fig 6H). Osteonectin, osteocalcin and VEGF antibodies were significantly higher in group T than in group C (p ϕ,δ,* = 0.0001), but no differences were found in TGF-β antibody (p ˃ 0.05) for osteoblastic immunoreactivity. T = Teriparatide group, C = Control group. reported that teriparatide reduced bone loss after implantation and caused improved osseointegration. Marques et al. 12 and Bashutski et al. 35 examined the effects of intermittent teriparatide on periodontitis, and reported that teriparatide prevented bone loss due to periodontitis and allowed a better healing of the defect. The effects of teriparatide on tooth movement during orthodontic therapy were evaluated by Salazar et al, 36 who examined the effects of subcutaneous intermittent teriparatide (30 μg/kg) on the tooth movement of the maxillary first molars in ovariectomized rats for 90 days, and concluded that the rate of orthodontically induced tooth movement was greater in rats treated with teriparatide, because of its activation effects on osteoblasts.

Immunohistochemical evaluation
They 36 reported that there was more tooth movement when injected teriparatide, without a significant increase in osteoclast number, since teriparatide normalized bone density.
In another study, Soma et al. 37 reported that the continuous application of 10 μg/kg teriparatide for 7 days could accelerate tooth movement. Soma et al. 38  Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion 22 Studies investigating the effects of teriparatide used a variety of methods, including histologic, 12,13 histomorphometric, 34 radiological 11,12 and tomographic 11 methods. However, to the best of our knowledge, no study has yet investigated the effects of teriparatide on bone formation by the immunohistochemical method, which may provide reliable and reproducible results, because of the targeting of a smaller and more specific structure of cells. According to the present results, the immunoreactivity of the osteoblasts for osteonectin, osteocalcin and VEGF antibodies in the suture region was higher in group T than in group C (p = 0.0001). Histologically, the excess of the number of osteoblasts in the suture zone in group T was consistent with the finding that the amount of newly formed bone in the suture region is higher than that in group C. In group T, the increase in the number of osteoblasts resulting from the increase in osteoblast stimulation was similar to previous studies. 1,6,22,39 Birlik et al. 13 evaluated the effects of sex steroids, namely, testosterone and oestrogen, on bone formation following a midpalatal suture expansion in rats. The female experimental group was given 17b-estradiol at a dose of 0.1 mg/kg daily, and the male experimental group was administered testosterone at a dose of 4 mg/kg intramuscularly throughout the expansion and retention periods of 7 and 5 days, respectively. Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion 23 The authors 13 detected a strong immunoreactivity of VEGF, TGF-β, osteocalcin and osteonectin in the osteoblasts and in the connective tissues of sutures. In the current study, indirect immunohistochemical staining with osteocalcin, osteonectin and VEGF showed a high immunoreactivity in the osteoblasts, but the slight increase in TGF-β immunoreactivity in group T did not reach significance. However, a full comparison was not made because of the differences in the study designs of Birlik et al. 13 (orchiectomised and ovariectomised rats) and the present study, and in the retention times.
After the maxillary expansion procedures, different retention periods such as 7, 3,39 10 40 and 12 days 41 were used. However, no consensus has been made in the literature on the retention period required after RME. The response after orthodontic force is about 30-40 hours in humans and 6 hours in rats, and the basic mechanisms in these changes are similar to humans. 42 Saito  Çamili Y, Malkoç S, Taşlıdere A, İleri Z, Guler OC -Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion 24 appliance of Saito and Shimizu 3 was used to provide sutural separation with incisor teeth support and to prevent difficulties during the placement of wires around the spring due to the small volume of the rat maxilla. Furthermore, we used systemically administration of teriparatide 27,28 rather than a local injection, as suggested in the literature, during orthodontic treatment, because of the possibility of a direct effect on the osteoblasts. 43 Local administration was reported to fail because it could not specifically target the osteoblastic activity. 3,39 The ease of application, the vascular structure of the maxilla, and the wide use of systemic administration in the literature makes it a more effective method than local injection.
However, systemic application was claimed to be involved in jaw bone necrosis. 44 In addition, systemic usage was found to cause bone tumors by using a 30 μg/kg daily dosage for 2-6 months in rats. 45 In the current study, no abnormalities from bone analysis were detected. Side effects such as headache,

Funding: This work was supported by a research grant from İnönü University, Scientific
Research Committee (2013/149).