Materials and methods

The animal experimental protocol in this study was approved by by the ethics committee for animal experiments at the Nihon University School of Dentistry at Matsudo (approval number AP14MD008) in Japan. A total of 50 male 11-week-old Wistar rats (body weight, 300 ± 30 g; Sankyo-Lab Service, Tokyo, Japan) were used for the experiments. The animals were maintained in the animal center of the School of Dentistry in separate cages in a 12-hour light and dark environment at a constant temperature of 23°C and were given food and water ad libitum. The health status of each rat was evaluated based on daily body weight monitoring for a week before the start of the experiments. The rats were randomly assigned to 2 groups (via simple randomization); tooth movement was applied by 10 g of orthodontic force to the maxillary first molar (TM) in the control group, and the TM plus MOPs of the cortical plate (TM + MOPs). Twenty-five rats in each group were used. We calculated our sample size based on the method of Cheung et al, and the number of subjects was based on our pilot study. The experiments were performed for 14 days ( Fig 1 ).

Experimental time schedule for each group.

In each group, the animals were anesthetized using an intraperitoneal injection of 3 mixed anesthetics, hydrochloric acid medetomidine (domitoru), mitazoramu, and butorphanol tartrate (betorufamu), (0.15 mg/kg body weight) for the application of orthodontic devices. Experimental tooth movement was induced using the method of Asano et al with a closed-coil spring (wire size, 0.005 in; diameter, 0.083 in; Accurate, Tokyo, Japan) ligated to the maxillary left first molar with a 0.008-in stainless steel ligature wire (Tomy International, Tokyo, Japan). The other side of the coil spring was also ligated, with holes in the maxillary incisors drilled laterally slightly higher than the gingival papilla using a round bur, applying the same ligature wire. The first molar was moved mesially by the closed-coil spring with a force of 10 g. The period of the experiment was 14 days ( Fig 2 , A ).

The method used for experimental tooth movement: A, the maxillary first molar was moved mesially by a closed-coil spring with a force of 10 g; B, schematic illustration showing the 3 shallow perforations (0.25 mm in diameter and depth) created 5 mm mesial to the first molar; C, the area of investigation ( shaded box ) was in the mesial aspect of the distal root of the first molar. The periodontal tissues in the pressure area were one-quarter of the mesial area facing the distal-buccal root, as determined when linked with the center of the distal-buccal root and the mesial-buccal root of the first molar. The large arrow indicates the direction of the force. Observations of the positive cells were performed in a 300-μm section ( shaded box ) of the root from the bifurcation surface on the mesial side, which is the pressure side during tooth movement.

MOPs were induced using the method of Teixeira et al. In the TM + MOPs group, a soft tissue flap was raised around the first molar. The flaps were sealed with cyanoacrylate tissue adhesive (Vetbond; 3M, St Paul, Minn). Using a round bur and hand piece, 3 perforations were delivered to the buccal alveolar bone 5 mm mesial to the left first molar. The diameter and depth of each perforation was 0.25 ± 0.005 mm, which we measured using microcomputed tomography (μCT) ( Fig 2 , B ).

To quantify the tooth movement, the distance between the enamel on the most distal aspect of the first molar, and the mesial aspect of the second molar was measured using μCT in each animal. A quantitative imageology analysis of the amount of OTM was performed using an in-vivo μCT system (Rigaku-μCT, Tokyo, Japan). Rat molars were scanned using μCT with an x-ray source of 90 kV/88 μA at 0, 1, 4, 7, 10, and 14 days. After being deeply anesthetized with intraperitoneally injected sodium pentobarbital (35 mg/kg), each rat was placed on the object stage, and imaging was performed over a full 360° rotation with an exposure time of 17 seconds. An isotropic resolution of 30 × 30 × 30 μm voxel size was selected, which displayed the microstructure of the interdental distances between the first and second molars. The original 3-dimensional images were displayed, and the data were analyzed with the I-View software program (J. Morita, Kyoto, Japan).

Furthermore, to evaluate the differences in the bone volume/tissue volume (BV/TV) ratio and bone mineral density (BMD) between the TM and TM + MOPs groups, the region of interest was delineated to encompass the alveolar bone region surrounding the first molar, with limits at 1 mm mesial of the first molar to the mesial aspect of the second molar. Volumetric quantification of the root volume of the first molar was performed in accordance with the previously established protocol of Furfaro et al. After highlighting the root structure, reconstruction of slices produced a 3-dimensional representation of the root structure so that 3 buccal roots and 2 palatal roots could be digitally resected and their volumes analyzed individually. A global threshold of 80 was applied to all scans to extract physiologically accurate representations of the alveolar bone phase.

The experimental period was set at 14 days after tooth movement began. The animals were deeply anesthetized by 3 mixed anesthetics and then transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer; then the maxilla was immediately dissected and immersed in the same fixative for 18 hours at 4°C. The specimens were decalcified in 10% disodium ethylenediamine tetraacetic acid (pH 7.4) solution for 4 weeks, and the decalcified specimens were then dehydrated through a graded series of ethanol washes and embedded in paraffin using the usual methods for preparation. Each sample was sliced into 4-μm thick sections continuous in the horizontal direction and then prepared for hematoxylin and eosin, immunohistochemical staining, and the TUNEL assay. The periodontal tissues in the mesial part of the distal-buccal root of a maxillary first molar were observed. The animals in which the tooth did not move were assigned to the control group ( Fig 2 , C ).

For immunohistochemical staining, the sections were deparaffinized, and the endogenous peroxidase activities were quenched by incubation in 0.5% hydrogen peroxide in methanol for 30 minutes at room temperature. After washing in tris-buffered saline solution, the sections were incubated with TNF-α antibodies (polyclonal goat, working dilution, 1:100; R&D Systems, Minneapolis, Minn) and PCNA antibodies (PC10, mouse monoclonal, working dilution, 1:100; Cell Signal Technology, Tokyo, Japan) for 18 hours at 4°C.

TNF-α was stained using the Histofine Simple Stain MAX-Po (G), and PCNA was the Histofine Simple Stain MAX-Po (M) kit (Nichirei Biosciences, Tokyo, Japan) in accordance with the manufacturer’s protocol. The sections were rinsed with tris-buffered saline solution, and the final color reactions were performed using the 3, 3′-diaminobenzidine tetra-hydrochloride substrate reagent (DAB). The sections were then counterstained with Mayer’s hematoxylin. As immunohistochemical controls, several sections were incubated with either nonimmune rabbit IgG or 0.01 M phosphate-buffered saline solution instead of the primary antibody. Negative reactivity was observed for the controls.

Apoptosis was detected by the TUNEL assay using the TACS 2TdT-DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, Md). The paraffin sections (4 μm) were incubated with proteinase K (Trevigen), diluted 1:200 at 37°C for 15 minutes, washed with deionized water, incubated with 0.5% hydrogen peroxide for 5 minutes, and then washed with deionized water again. For the positive control, the sections were incubated with DNase I at 37°C for 30 minutes. All sections were incubated with terminal deoxynucleotide transferase and Biotin-dUTP at 37°C for 1 hour (negative control sections were not incubated with the terminal deoxynucleotide transferase enzyme). After washing, the sections were incubated in strep-HRP solution at 37°C for 10 minutes. After washing, DAB was added to demarcate the apoptotic cells. The sections were counterstained with 1% methyl green and finally dehydrated and mounted.

The ratio of TNF-α, PCNA, or TUNEL positive cells was calculated as the number of positive cells in all cells observed in both the pressure and tension sides at each time period, using the following formulae.

• 1.

  Ratio of TNF-α positive cells = (number of PCNA positive cells/number of all cells) × 100.

• 2.

  Ratio of PCNA positive cells = (number of TUNEL positive cells/number of all cells) × 100.

• 3.

  Ratio of TUNEL positive cells = (number of TUNEL positive cells/number of all cells) × 100.

Three sections were counted for both observed areas at each time period.