Material and methods

All animal procedures and the study protocol were approved by the Veterinary Administration of the Republic of Slovenia (number 323-02-234/2005/2) and are in compliance with the guiding principles in the “Care and Use of Animals.” The study was performed with 48 male Wistar rats (320-340 g, 13-14 weeks old). The animals were housed in groups of 4 in polycarbonate cages under normal laboratory conditions at constant temperature (24°C-25°C) and humidity, with a 12-hour circadian cycle. They were fed with soaked standard laboratory rat chow diet (Krka, Novo mesto, Slovenia) and water ad libitum. The rat chow was soaked in water to facilitate food intake that was impaired by the orthodontic appliance. General anesthesia was used for placing the orthodontic appliance. To ensure general anesthesia, a mixture of 3 anesthetics was used. The anesthetics were injected intraperitoneally: ketamine at 50 mg per kilogram ⁻¹ of body weight (Bioketan, Vetoquinol Biowet, Gorzów Wielkopolski, Poland), medetomidin hydrochloride at 67 μg per kilogram ⁻¹ of body weight (Domitor, Pfizer, Brooklyn, NY), and thiopental at 3 mg per kilogram ⁻¹ of body weight (Tiopental, Pliva, Zagreb, Croatia).

The orthodontic appliance consisted of a superelastic closed-coil spring (25 cN; wire diameter, 0.15 mm; GAC International, Bohemia, NY), placed between the maxillary left first molar and the incisors. The superelasticity of the coil spring ensured a constant force (25 cN) during the whole time of coil-spring activation. The closed-coil spring was attached to the maxillary left first molar with a stainless steel ligature wire (diameter, 0.25 mm; Dentaurum, Ispringen, Germany) and to the incisors by a surgical steel wire (4-0, multifilament, W310 , Ethicon; Johnson & Johnson, New Brunswick, NJ). To improve the fixation of the appliance, a 0.5-mm hole was made by using a hard metal burr (HM 1, 204, 005, Meisinger, Neuss, Germany). The hole was drilled from the distal surfaces of the incisors, through the teeth, and perpendicular to the longitudinal axis of the incisor at the gingival level. The steel wire was inserted through the hole and bonded to the proximal surface of the right incisor.

The animals were divided into 3 groups, 1 control and 2 appliance groups (appliance-only and cetirizine). In the control group (n = 16), no orthodontic appliance was placed, and each animal received 0.1 mL of saline solution daily. In the appliance-only group (n = 16), each animal was fitted with an orthodontic appliance and received 0.1 mL of saline solution daily. In the cetirizine group (n = 16), each animal was fitted with an orthodontic appliance and received 3 mg of kilogram ⁻¹ of cetirizine (Krka) daily. All animals received saline solution or cetirizine at approximately the same time for 42 days. The orthodontic appliance was placed under general anesthesia at the beginning of the study and replaced to the correct position every 7 days to ensure its proper activation, and consequently a constant force was exerted on the teeth. On day 42, all animals were killed.

The distance between the most mesial point of the maxillary left first molar and the most palatal point of the ipsilateral incisor at the gingival level was measured in all animals. Measurements were made weekly with a digitronic caliper with an accuracy of ± 0.01 mm (Wilson & Wolpert, Utrecht, The Netherlands) while the animals were anesthetized. All measurements were made twice by 2 investigators (A.M., Š.S.) independently within a few minutes. Tooth movement was calculated by subtracting the distance between the teeth on each day of measurement (days 7, 14, 21, 28, 35, and 42) from the distance between the teeth measured the previous week.

Cathepsin K gene expression level was used to determine osteoclast activity, and osteocalcin gene expression level was used to determine osteoblast activity. On day 42, 8 animals from each of the 3 groups were killed with an intraperitoneal injection of anesthetics and carbon dioxide. Tissue samples of alveolar bone, with all 3 molars and their PDLs, were excised and frozen in liquid nitrogen and mechanically powdered. The total ribonucleic acid (RNA) content from 120 ± 30 mg of each powdered sample was isolated by using TRIzol reagent (Invitrogen, Carlsbad, Calif), according to the manufacturer’s instructions. RNA concentrations and quality were determined by the Agilent 2100 Bioanalyzer with an RNA microfluidics chip (Agilent, Santa Clara, Calif).

To prepare primary cDNA, 10 ng of total RNA was used along with a high-capacity cDNA archive kit, with the addition of RNase inhibitor (Applied Biosystems, Foster City, Calif). The reaction was conducted according to the manufacturer’s recommendations at a final volume of 100 μL. It proceeded at 25°C for 10 minutes and 37°C for 2 hours, and was stopped by inactivation at 99°C for 5 minutes. Oligonucleotides for cathepsin K and osteocalcin were chosen from predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems, Rn00580723_m1, Rn01455285_g1, respectively). Thermal cycling comprised initial steps at 50°C for 2 minutes and at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. A standard curve was constructed with serial dilutions of a mix of a few samples. The cDNA was amplified and quantified by using a sequence detection system (SDS 7000, Applied Biosystems, Foster City, Calif). To exclude variations from different inputs of total mRNA to the reaction, data on cathepsin K and osteocalcin were normalized to an internal housekeeping gene, GAPDH, for which data was obtained by using TaqMan GAPDH predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems, Rn01462662_g1). All reactions for standard samples and for samples from all 3 groups (control, appliance-only, and cetirizine) were performed in duplicate. The data were averaged from the values obtained in each reaction.

Bone histomorphometry was used to determine alveolar bone volume density, osteoclast volume density, and osteoblast volume density in all 3 groups. On day 42, the remaining 8 animals from each group were killed by intraperitoneal injection of anesthetics and carbon dioxide. Tissue samples of the maxilla containing all 3 molars were taken. Tissue specimens were prepared as described. Histomorphometry was performed by using a point-counting method. For this purpose, the stereologic cycloid grid system incorporated into the ocular of a light microscope (BX-60, Olympus, Tokyo, Japan) was used. All histomorphometric measurements and calculations were made according to the standard nomenclature. The alveolar bone area density, expressed as the percentage of the tissue area consisting of tooth, PDL, connective tissue, and bone marrow spaces (B.Ar/T.Ar), was determined at 10-times magnification. Osteoclast and osteoblast area densities, defined as the alveolar bone area covered with either osteoclasts (multi-nucleated cells at the alveolar bone surface) or osteoblasts (“round” cells at the alveolar bone surface) as a fraction of the total alveolar bone area (Oc.Ar/B.Ar or Ob.Ar/B.Ar), were determined at 40-times magnification. The borders of the tissue area examined were determined as described by Sprogar et al. Figure 1 shows the examined area. Since 20 sections were examined from each part (apical, middle, and cervical) of the root of the maxillary left first molar, the 2-dimensional term “area density” was extrapolated to the 3-dimensional term “volume density.” Alveolar bone area density, osteoclast area density, and osteoblast area density were extrapolated to alveolar bone volume density (BV/TV), osteoclast volume density (Oc.V/BV), and osteoblast volume density (Ob.V/BV).

A horizontal section through the mid third of the roots of all 3 maxillary left molars at 10-times magnification. The alveolar bone volume density (BV/TV) around all 5 roots of the maxillary left first molar ( A + B ) and the osteoclast and osteoblast volume densities (Oc.V/BV and Ob.V/BV) around the mesial root of the maxillary left first molar ( B ) were determined histomorphometrically.