Study design and experimental animals
A total of 90 male C57BL/6N wild-type mice (age: 8–10 weeks, Charles River, Fig. 5a) were randomly assigned to one of two different orthodontic procedures to induce experimental orthodontic tooth movement (OTM). 45 C57BL/6 N mice received a halved NiTi coil spring (10–000-26, Sentalloy, GAC International, Fig. 5b) and 45 C57BL/6 N mice an elastic band (Inwaria, thread elastic Ø 0.3 mm; Fig. 5c), respectively, for anterior movement of the first upper left molar. The contralateral untreated jaw side served as a non-force internal control (split-mouth model) and all measurements were performed at both jaw sides. 15 mice were included in each group. For micro-CT and histological analyses seven animals (n = 7) per time point and for RT-qPCR analyses eight animals per time point (n = 8) were examined. After 3, 7 or 12 days of OTM, the mice were euthanized according to legal guidelines. Sample sizes were based on a previous study41 using a similar experimental setup to induce orthodontic tooth movement in rats by means of a NiTi coil spring as well as a similar technique of retrieving samples and performing RT-qPCR analyses, which proved to provide sufficient study power, while minimizing animal suffering.
The animal experiments were carried out with the approval of the responsible authorities (AZ: 55.2-2532-2-567) and in compliance with the German Animal Protection Act. In order to avoid unnecessary suffering of the animals, corresponding termination criteria were predefined and animal condition as well as gross body weight monitored daily. The animals were kept in a conventional S1 animal laboratory at the University of Regensburg. The laboratory maintains a temperature of 21 °C and a relative humidity of 55% with a 12-h light–dark rhythm with the light phase between 7:00 h and 19:00 h. The experimental animals were kept in groups of six in transparent polycarbonate cages with a metal grid cover. The mice had ad libitum access to distilled water in plastic bottles, which was renewed twice a week, and to a standard diet (V1535, ssniff) with feed pellets softened to mash from the beginning of orthodontic treatment and placed in a feeding trough inside the cage. After an acclimatisation phase of at least one week after delivery, the experiment was started so that the baseline age of the animals was 9–10 weeks with an average weight of 24.4 g (SD 1.2 g).
As anaesthetic, xylazine/ketamine was used in the ratio 1: 3, which was diluted 1: 5 with physiological saline. Animals receiving a feather were injected intraperitoneally with 0.008 ml of the dilution per gram of body weight. The animals treated with an elastic band received standardized 0.1 ml of the dilution, because they had to be anesthetized for a much shorter period of time.
For treatment, animals were positioned on their back in a custom-made apparatus (Fig. 5a), which was modified from a previously used system in rats16. The upper jaw was immobilised by an anatomical probe spanning across the palate between the upper incisors and molars, which is connected to the platform of the apparatus by rubber bands, thus pressing the head firmly to the platform. To enable easy access to the oral cavity for interventions, the mandible and tongue were retracted from the immobilised upper jaw until an adequate mouth opening was achieved by means of a wire loop around the mandibular anterior teeth and tongue, fixated via a connecting wire with an inserted coil spring as stressbreaker in the appropriate tensed state at a vertical beam at the back of the platform.
Experimental tooth movement with a NiTi coil spring
A NiTi coil spring with the lowest commercially available force level of 0.25 N (10-000-26, Sentalloy, GAC International) and a length of 3 mm was cut in half, as due to the limited available space between the upper mouse molars and incisors a tensioning of the spring would otherwise not have been possible. Subsequently, two orthodontic stainless steel auxillary wires (Ø 0.08 mm) were threaded directly into the spring, as described previously16 (Fig. 5b, Inset). To ensure a uniform force level of about 0.35 N by spring extension as recommended by Taddei et al.42, which was quantified and adjusted upon insertion with a calibrated orthodontic spring balance (Correx, small model, Haag-Streit AG, Köniz, Switzerland) as described before41, care was taken to ensure that there are always exactly four coils between the two wires. The spring was attached to the neck of the first left upper molars with a wire loop initially threaded through the proximal space between the first and second upper molars from palatally (Supplementary Fig. 1a) enclosing both the first molar and the end of the spring with its inactive coils (Supplementary Fig. 1b). Then the spring was stretched to the upper incisors and the auxillary wire inserted at the anterior end encompassing the terminal inactive coils was threaded through the approximal space of the maxillary incisors (Supplementary Fig. 1c), passed around the two incisors, then guided through the inactive anterior terminal coils of the spring at the neck of the incisors, and again threaded through the approximal space of the incisors (Supplementary Fig. 1d). There, the two wire ends could be briefly attached to the holding apparatus with an adhesive tape in order to avoid a change in position. The final anterior attachment to the incisors was achieved by means of layer of flowable composite over the wire loop and the anterior teeth. For this purpose, the surface of the incisors was etched with 37% phosphoric acid (i-Bond Etch 20 gel, Heraeus Kulzer) for 120 s, rinsed for 15 s and dried with an air syringe for 15 s. Subsequently, Transbond XT primer (3 M Unitek) was applied by means of a microbrush and light-cured for 20 s. Subsequently, a thin flowable composite coating (Tetric EvoFlow A3, Ivoclar Vivadent) was applied to the wire loop and the tooth, shaped with a dental probe and light-cured for 40 s. A small oral extension of the coating encompassing the terminal inactive end of the coil spring was formed to protect the delicate transition between the composite coating and the NiTi spring during mastication. Afterwards the auxillary wire ends fixated with the adhesive tape could be loosened, twisted around each other (Supplementary Fig. 1e), shortened, bent laterally and covered again with some additional flowable composite to avoid injuries from a sharp wire end (Supplementary Fig. 1f, Fig. 5a). The mandibular anterior teeth were cut at papillary level once upon appliance insertion with a diamond-plated cutting disc at 10,000 rpm to prevent damage to the spring by the lower incisors during mastication.
Experimental tooth movement with an elastic band
For the investigation of the Waldo/Rothblatt method, an elastic band with a diameter of 0.3 mm (Inwaria) was used (Fig. 5c, Inset), which was the largest diameter insertable in the proximal space for maximal experimental tooth movement. First, the proximal space between the first and second upper molars was pre-expanded with an orthodontic auxillary wire (Ø 0.08 mm), then the elastic band was inserted using two Mosquito clamps (straight, with teeth) and then shortened accordingly at both sides (Fig. 5c).
µCT analyses for evaluation of periodontal bone properties/loss and tooth movement
Seven animals per experimental group and timepoint were perfused with 5% formaldehyde and upper jaws disected and stored overnight in 5% formaldehyde. Jaws were then transferred to 0.1% formaldehyde for long-time storage until µCT measurements were performed, which were carried out at the OTH Regensburg with the device Phoenix vltomelxs 240/180 (GE Sensing & Inspection Technologies), with a 180 kV-NF tube using the following settings: Voxelsize: 10 μm, Images: 1800, Timing: 333 ms, Voltage: 50 kV, Current: 750 μA, Fastscan: Scan duration 10 min. Image reconstruction and evaluation was performed with the software Volume Graphics—VG Studio Max (Volume Graphics GmbH). These were all performed within a sagittal layer plane, aligned perpendicular to the occlusal plane, parallel to the palatal suture and placed through the middle of the first molar at the level of the cemento–enamel junction (CEJ) in order to allow reproducible measurements. To determine the properties of the alveolar bone, an interradicular cube was created, by using the “regions of interest” (ROI) function. The edge lengths of the inserted cube were 0.35 × 0.35 × 0.35 mm (height × width × depth, Supplemental Fig. 2). These specified values were exported as a file, saved and imported for all other µCT images to be measured and adopted as a measurement variable. As due to minimally varying tooth root morphology of the first upper molar from animal to animal an exact landmark-based approach to place the cubical region of interest was not possible, we set the cubical region of interest manually-visually in the central interradicular region 0.2 mm below the furcation taking special care not to transgress into other non-bony structures such as the tooth roots. After extracting the data, the cube could be viewed and evaluated as a volume. Periodontal bone loss at the first molar was measured distally along the root surface as an increase of the distance between the cemento–enamel junction and the alveolar limbus (Supplemental Fig. 3a). To quantify the extent of orthodontic tooth movement, the smallest distance between the crowns of the first and second upper molar was measured with the vernier caliper function of the software (Supplemental Fig. 3b).
Preparation of histological samples
After µCT analyses jaws were divided into the control and orthodontically-treated (OTM) side and demineralised in Tris-buffered ethylene diamine tetra-acetic (EDTA) solution (10%, pH = 7.4)43 for eight weeks at room temperature. Jaws were then embedded in paraffin and cut in sagittal-oblique sections of the tooth-bearing alveolar process of approximately 5 µm using a rotating microtome (HM350, Microm International). All sections were mounted onto Superfrost glass slides (SuperFrost Plus).
HE staining for evaluation of orthodontically induced inflammatory root resorptions
Sections were deparaffinised at 60 °C for 30 min and directly transferred to xylene (9713.2, Carl-Roth) for 20 min. Sections were hydrogenated by a descending series of alcohol consisting of twice 100% ethanol, 96% ethanol, 70% ethanol and H2Odd for 10 min each. This was followed by staining of the cell nuclei with Mayer haematoxylin solution (1.07961.0500, Sigma-Aldrich) for 10 min. Afterwards, slides were incubated under running, warm water for 5 min. This was followed by counterstaining with freshly prepared eosin G solution 0.5% (X883.2, Carl-Roth) for one minute. Thereafter, the sections were rinsed under warm tap water and dehydrated by the ascending series of alcohol, with dips in the 70% and 96% ethanol sections only briefly to avoid discoloration. After a residence time of at least 20 min in xylene, the coverslips were applied with entellan (1.07961.0500, Merck). The stained histological sections were photographed and digitized under the microscope (Keyence BZ-X800, Neu-Isenburg, Germany, Supplemental Fig. 4). The relative extent of orthodontically induced inflammatory root resorptions (OIIRR) at the mesio-buccal root of the first upper molar was quantified with the software ImageJ (Ver.147, National Institutes of Health, USA) as total resorption area to total root area (between the CEJ and the apex of the furcation) as described before44.
TRAP (tartrate-resistant acid phosphatase) staining for evaluation of osteoclastogenesis
Paraffin sections were incubated overnight at 37 °C and then hydrogenated via a descending alcohol series (compare HE staining) and rinsed in H2Odd. Slides were placed for 10 min at room temperature in a previously freshly prepared TRAP buffer consisting of 1.64 g sodium acetate (6773.1, Carl-Roth) and 23 g of di-sodium tartrate dihydrate (T110.1, Carl-Roth) to 500 ml of H2Odd. pH was adjusted to 5 with HCl. Sections were then placed in a freshly prepared staining solution consisting of 40 mg of Naphtol AS-MX Phosphate Disodium Salt (N5000, Sigma-Aldrich), 4 ml of N,N-dimethylformamide (D4551, Sigma-Aldrich), 240 mg of Fast Red Violet LB Salt (F3381, Sigma-Aldrich), 2 ml Triton X-100 (T9284, Sigma-Aldrich) and 200 ml of the previously prepared TRAP buffer. The sections were incubated at 37 °C for two hours, then rinsed in H2Odd and counterstained for 3 min with filtered Mayer’s haematoxylin solution (51275, Sigma-Aldrich) at room temperature. Subsequently, the sections were covered immediately with Aquatex (1085620050, Merck). Stained histological sections were photographed under the microscope (Keyence BZ-X800, Neu-Isenburg, Germany, Supplemental Fig. 5). The relative extent of the TRAP-positive area corresponding to osteoclastogenesis and osteoclast activity within the periodontal ligament and adjacent bone and tooth surfaces at the mesio-buccal root of the first upper molar was quantified with the software ImageJ (Ver.147, National Institutes of Health, USA) using the “Color Threshold” function (Hue 189-255, Saturation 50 255, Brightness 0-203, Method: Default) as TRAP-positive area to total root area (area between the CEJ and the apex of the furcation) as described before44.
RNA isolation, reverse transcription and RT-qPCR analysis
A cuboid tissue sample of defined extension was prepared of eight animals per experimental group and timepoint with a sterile cutter by macrodissection, containing the first upper left or right molar without the clinical crowns with adjacent periodontal tissue and alveolar bone as described in Kirschneck et al.41, immediately transferred into liquid nitrogen and stored at − 80 °C until RNA isolation could be performed using the RNeasy Mini Kit (74104, Qiagen) according to the manufacturer’s instructions. Before RNA extraction, samples were refrozen in liquid nitrogen and then pulverized in a bone mill (Retsch). The recovered RNA was finally quantified and checked for purity as described before41 with a nano-photometer (Implen). For cDNA synthesis RNA was added to nuclease free water depending on the concentration. 11 μl of this solution was added to 9 μl of a previously prepared master mix. This consisted of random hexamer primer (0.1 nmol, 1 μl, SO142, Life Technologies), oligo-dT18 primer (0.1 nmol, 1 μl, SO131, Life Technologies), 5 × M-MLV Buffer (4 μl, M1705, Promega), dNTP mixture (40 nmol, 1 μl–10 nmol/dNTP, L785.2, Carl Roth), reverse transcriptase (200 U, 1 μl, M1705, Promega) and RNase inhibitor (40 U, 1 μl, EO0381, Life Technologies). The samples were incubated for 1 h at 37 °C, followed by an incubation at 95 °C for 2 min to inactivate the reverse transcriptase. Subsequently, the cDNA was stored at − 20 °C until further processing. RT-qPCR amplification reaction was carried out as described before41 in a Mastercycler ep realplex thermocycler (Eppendorf) with 96-well PCR plates (712282, Biozym Scientific) and adhesive optical seal film (712350, Biozym Scientific). SYBR Green JumpStart Taq ReadyMix (7.5 μl, S4438, Sigma-Aldrich), the respective primer pair (0.75 μl per primer; Table 2) and 1.5 μl of the corresponding cDNA were filled up to a total volume of 15 μl with nuclease-free water (T143, Carl-Roth). Cq values were identified as second derivative maximum of the fluorescence signal curve employing the software realplex (version 2.2, EppendorfAG, CalQPlex algorithm, Automatic Baseline, Drift Correction On). Evaluation of the RT-qPCRs was carried out by the method of relative quantification as described before41,44,45. Briefly, the expression of the target genes was related to that of the reference gene Eef1a1 and defined as 2−ΔCq with ΔCq = Cq target gene − Cq (Eef1a1). Prior to statistical analysis of relative gene expression data, all data values were divided by the respective arithmetic mean of the not-orthodontically-treated jaw side at each timepoint for each treatment to obtain normalized data values relative to these controls, set to 1.
Data analysis and statistics
Statistical evaluation was performed with the program GraphPad Prism (Version 8.00 for Windows, GraphPad Software). Normal distribution was tested with a Shapiro–Wilk test. Depending on distribution of data either welch-corrected ANOVAs followed by Games-Howell multiple comparison test or ordinary ANOVAs followed by Holm Sidak’s multiple comparison tests were performed. Extent of tooth movement was analysed using Kruskal–Wallis test followed by Dunn’s multiple comparison test. The graphs show the individual data values as well as the mean ± standard error of the mean. Significance was assumed at p ≤ 0.05.