Pantoprazole, a proton pump inhibitor, increases orthodontic tooth movement in rats

Introduction

Proton pump inhibitors (PPIs) such as pantoprazole act by irreversibly binding to H+/K+ ATPase ion-exchanger or the proton pump located in the apical surface of acid secreting parietal cells in the stomach (1). PPIs are regarded as remarkably safe and effective agents for treatment of upper gastrointestinal disorders (2). Although these drugs are one of the most frequently prescribed medications, have recently raised concern that taking them, particularly at high doses, may result in decreased calcium absorption, hypocalcaemia, and therefore osteoporosis (3, 4). Two population-based epidemiological studies in Denmark and UK have supported this hypothesis (5, 6).

The alveolar bone surrounds dental tissues and is constantly undergoing remodeling which is considered to be the basis of the orthodontic tooth movement (OTM) (7, 8). Application of mechanical forces during orthodontic treatment induces modifications in the remodeling of the supporting alveolar bone, resulting in a predominance of bone resorption in the pressure side, and an advantage              of bone formation in the tension side (7). Osteoclasts elicit bone resorption and are generated from their precursors following force application. Some of these precursor cells already exist in the periodontal ligament, but others are recruited to the pressure side within 2 to 3 days. Differentiation and polarization of osteoclasts follow, which ultimately

Table1. Timetable for drug administrations in each study group

OTM: Orthodontic tooth movement; App: Appliance; Panto/Veh: Pantoprazole/vehichle administration, BDE: Bone densitometry evaluation

lead to resorption of osseous tissue. Osteoblasts are responsible for bone formation and are derived from local precursor cells in the tension side, which differentiate into mature osteoblastic cells that produce osteoid. This is ensued by gradual mineralization and generation of bone (9). It has been shown that local and systemic factors are able to reactivate osteoclasts which dissolve the minerals through secretion of hydrogen ions and degrade the organic matrix by liberating proteolytic enzymes. We have previously reported the effect of renal insufficiency, hypothyroidism, and cholestasis on the rate of OTM; therefore it may be logical to suggest that any factor causing systematic alteration of bone metabolism such as hypocalcemia, can potentially increase the rate of OTM (10-13). Rat models have been used in various investigations and their result was compared and generalized to similar human events (14-16). This study was conducted to determine whether PPIs could alter the rate of orthodontic tooth movement in rats.

 Materials and Methods

Study design

The present investigation was designed as a time-course study and our sample consisted of 72 male Sprague-Dawley rats (nine weeks old) weighing from 200 to 250 g, which were divided into six groups of 12 animals each. Their diet consisted of soft laboratory food in order to facilitate eating after placement of the appliances and to reduce the possibility of the animals displacing their orthodontic devices. The humidity and temperature of the caging rooms were controlled and maintained with a 12 hr light and dark cycle. All experiment conditions and procedures strictly adhered to the guidelines described by the US National Institute of Health (NIH publication no. 85.23, revised 1985 for the Care and Use of Laboratory Animals and the study protocol were approved by the ethics committee of our university. Each of the six study groups were further divided into two subgroups including eight test animals that received pantoprazole 200 mg/kg in 0.25 percent carboxymethyl cellulose (CMC) by a gastric tube (pantoprazole treated rats) and four rats that received only CMC (vehicle only treated rats) (17). A 2-week course of orthodontic treatment was performed for all animals, during which the rats were given their routine experimental agents (pantoprazole or vehicle). The six groups were as follows: 1) a group that received pantoprazol and appliance from the first day of the study, 2) a group that received pantoprazole one week before the appliance insertion, 3) a group that received pantoprazole two weeks before the appliance insertion, 4) a group that received pantoprazole three weeks before the appliance insertion, 5) a group that received pantoprazole four weeks before the appliance insertion, 6) a group that received pantoprazole five weeks before the appliance insertion. Table1 shows the time table for drug administration, appliance insertion, radiography, and date of sacrifice in each study group.

 Drug administration

Pantoprazole (Byc Gulden, Konstanz, Germany) at a dose of 200 mg/kg daily was dissolved in 0.25 percent sodium carboxymethyl cellulose (9004-32-4, Sigma-Aldrich, Saint Louis, MO, USA) and was administered via a fine gastric tube. In addition, 65 mg/kg intraperitoneal pentobarbital was used for general anesthesia and they were sacrificed by a 100 mg/kg pentobarbital overdose at the end of the study.

 Survival study and general toxicity

Assessment of mortality and general toxicity including edema, cachexia, alopecia, and mortality were carried out daily in all groups while the body weight was measured twice a week through the whole experiment period.

 Placement of orthodontic appliances

The orthodontic appliance design used in the present investigation was similar to that employed

 Figure 1. OTM measurements in six study groups: each group consisted of four rats comprising the vehicle-only group (receiving only vehicle) and eight rats constituting the pantoprazole-treated group who received pantoprazole + vehicle (*P<0.05; ** P<0.01)

 by Shirazi et al (10). Briefly, nickel-titanium closed coil springs (NiTi, 3M Unitek, Monrovia, Calif, Hi-tek, 0.006 × 0.022 inch) were cut at a length of 5 mm and placed between the upper right first molar and incisors of all treated and vehicle-only animals to provide an initial force of 60 g. Reactivation was not performed during the 2-week study period due to the "flat load-deflection curve" of these appliances (12, 13). Maximum anchorage in the anterior region was achieved through uniting the right and left incisors with composite resin, therefore warranting minimal distal movement of the right incisor.

Measurement of tooth movement

Animals were sacrificed 2 weeks after orthodontic treatment by intraperitoneal administration of 100 mg/kg sodium pentobarbital. After removal of the maxillae, special trays covering the right three molars were fabricated of acrylic (Acropars, Iran) and impressions were made using polisyloxane impression material (Spidex, Colthene, Swiss) followed by pouring with dental stone type IV (SH 074, Ernst Hinrichs GmbH, Germany). Measurements of the mesiodistal spaces between the distal surface of the first- and the mesial aspect of the second-molar were performed on the stone casts, using a standard millimeter inter-proximal gauge (10, 11). Ten days before drug administration all animals were controlled regarding their healthiness. The baseline OTM in 8 normal rats with no drug or vehicle administration (data not shown) was also measured.

 Bone densitometry evaluation

After a stable anesthesia with a dose of 65 mg/kg pentobarbital, a custom-made cephalostat was utilized to obtain lateral skull radiographs. These radiographs were obtained on occlusal fast films once a week (only in group 6), from the first to the 6^th week of the study. Radiographic conditions including exposure (10 mA at 50 kV), film-tube distance (50 cm), and film types were identical for all animals. Bone density was assessed at the lower border of the mandible, exactly beneath the first molar, employing optical densitometry (12, 18). Baseline optical density of 8 rats (without receiving any drug or vehicle) was measured 10 days before the main study (data not shown).

 Histological evaluation

To assess possible histologic changes due to drug administration and orthodontic treatment, the right hemimaxillae of all animals were surgically removed subsequent to sacrifice, and placed in ten percent formalin for 10 days. The specimens were decalcified in 5% formic acid for at least seven days, after washing with tap water. Routine sample processing was carried out and five micrometer mesiodistal sections were cut from paraffin blocks followed by hematoxylin and eosin staining of every fifth sections. The mesial roots of the first molars from the cementoenamel junction CEJ[Z1]  to the terminal point of the apex were analyzed on the slide with the largest root area. The microscopic observations were recorded for each case using an Olympus BX51 light microscope equipped with a digital camera (DP25, Olympus) and analysis software (DP2-BSW, Olympus). Osteoclasts were characterized as multinucleated cells with vacuolated eosinophilic to light basophilic cytoplasm, resided in Howship lacunae, and demonstrated ruffled borders when active. Osteoblastic activity was assessed by observation of surfaces with active bone formation surrounded by osteoid and plump basophilic, cuboidal osteoblasts. Measurements of periodontal ligament (PDL) width was performed in the widest area between the root- and bone-surfaces. Root resorption was determined by counting the number of resorptive lacunae, measurement of depth, and largest width of each of the craters. All parameters were recorded for both mesial and apical root aspects.

 Statistical analysis

The results are reported as mean ± SE. Statistical analysis of body weight changes and OTM data were performed by independent sample t-test while repeated measures of ANOVA was used for evaluation of optical densitometry. Group differences were calculated by post-hoc analysis using LSD test. For all the tests, differences with values of P < 0.05 were considered significant.

 Results

Survival study and general toxicity

After 5 weeks of the study, 2 rats died in the group receiving pantoprazole for 5 weeks before  the appliance insertion. Administration of pantoprazol at the used dosage did not induce any general toxicity including edema, cachexia, and

Figure 2. Microscopic view showing the periodontium (c: cementum; b: bone) of the mesial root in (A) a vehicle only rat with a low osteoclast count (hematoxylin and eosin staining; original magnification, ×400) and (B) an animal belonging to group F with a considerable number of osteoclasts (o) on the bone surface (original magnification, ×400)

 alopecia. No significant difference in weight gain or loss was found between the pantoprazole treated and vehicle-only groups (P<0.05).