All experimental procedures were reviewed and approved by the Saga University Animal Care and Use Committee (number: 26-051-0) and were performed in accordance with the regulations on animal experimentation at Saga University.
We used 6-week-old male Wistar rats (Jcl: Wistar, outbred, no genetic modification) weighing 198.8-212.2 g (Kyudo Corporation, Tosu, Japan). Animals were acclimated a week before surgery and were housed with food and water ad libitum under specific pathogen-free conditions. Three animals were bred in each cage. Fifteen animals were used for micro-CT and histological analyses, 3 animals were used for scanning electron microscopy (SEM), and another 3 animals were used for in vitro experiments. The total number of animals used was 21. Since at least 3 animals were required for each experiment and each timepoint and 5 timepoints were required to evaluate bone healing, the minimum number of animals was adopted, in consideration of animal ethics. All animals met the appropriate conditions because the species, sex, age, and weight of the animals were almost the same. In addition, no animals were excluded from the analysis during the experiments because no adverse events occurred. They were anaesthetized intraperitoneal injections with 3 mixed anaesthetic agents (0.4 mg/kg dexmedetomidine hydrochloride, 2.0 mg/kg midazolam and 5.0 mg/kg butorphanol tartrate) and subcutaneous anaesthesia to the head by 2% lidocaine with 1/80,000 epinephrine (Showa Yakuhin Kako, Tokyo, Japan). We adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and Japanese guidelines in conducting these animal experiments.
For ultrasonic osteotomy, VarioSurg3® with a titanium nitride coating H-SG1 tip (Blade width 0.6 mm) (NSK, Tochigi, Japan) was used with a driving frequency of 30 kHz and an output power of 17 kW (average) under saline irrigation. The tip was the most frequently used in clinical cases, and it could cut a calvarial bone defect in a few strokes. For conventional rotary osteotomy, we used a Surgic Pro® (NSK, Tochigi, Japan) with a round bur with a 0.8-mm diameter (Komet, SC, USA) at 40,000 rpm and 80 N/cm under saline irrigation. The round bur used was able to cut the bone defect set in the same way as the UOD tip with a few strokes.
Osteotomy design and operation procedure
After the head hair was shaved, an incision was made into the scalp under the local and general anaesthesia mentioned above. Before cutting, the cutting line of the osteotomy was marked with a pencil using a template made of resin. We utilized a craniotomy design consisting of pairs of cuts described by Anesi et al. .
For the bone healing evaluation by micro-computed tomography (CT) and histology, we made an 8 × 2-mm bone defect in the right calvaria with a UOD and a bone defect of the same size in the left calvaria with an ROD. During the ROD cut, we took care not to damage the dura with the instruments. A square 8 8-mm osteotomy was used for surface morphological analysis by SEM. The wounds were irrigated after bone cutting and closed by 4-0 silk threads. The cutting time for each appliance was recorded. After surgery, meloxicam (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) was intramuscularly injected at 0.2 mg/kg. After the surgery, the rats were given drinking water mixed with antibiotics (cefaclor monohydrate, 10 mg/kg body weight, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for two days. Specimens were collected immediately after the operation (0 days) and 1, 2, 3 and 4 weeks later for micro-CT three-dimensional bone morphology measurements and for histological evaluation by haematoxylin-eosin (HE) staining.
Animals were euthanized by deep anaesthesia at 0 days and 1, 2, 3 and 4 weeks after surgical osteotomy. Three rats at each time point, 15 animals in total, were used in the experiment. Three animals were bred in each cage, and upon evaluation, the animals in randomly selected cages were selected and analysed to minimise potential confounders. After euthanization, the whole calvaria was removed carefully with surgical scissors to avoid damaging the defective area. Tissue samples were fixed with 4% paraformaldehyde for 24 h and rinsed with 0.1 M phosphate buffered saline and then acquired images by micro-CT (MCT-CB130, Hitachi Medical Corporation, Tokyo, Japan: tube voltage; 60 kV, tube current; 100 µA) followed by three-dimensional bone morphometry using TRI/3D-BON software (Ratoc Engineering, Tokyo, Japan). The parameters measured were BV/TV, which indicates the bone volume in total tissues in the region of interest (ROI). The ROI had a size of 150×280×100 voxels centred on the bone defects. The voxel size was 26 µm. The evaluator performed the analysis without knowing whether the bone defect had been made with a UOD or an ROD.
After micro-CT evaluation, samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) tetrasodium tetrahydrate solution for one week, dehydrated through ascending alcohol concentrations, paraffin embedded and sectioned 8 µm thick. The sections were then dewaxed and processed for HE staining for light microscope analysis. The osteotomy gap width of the two native bone surfaces surrounding the osteotomy was evaluated in the section around the middle of the wound using ImageJ software . The distance was measured at 2 points, and the average was evaluated.
To determine the structural difference between the UOD cut and the ROD cut, we collected bones from 3 rats in each group just after ostectomy. Specimens were fixed with 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA) with 2.5% glutaraldehyde (Sigma-Aldrich, St Louis, MO, USA) in 0.2 M phosphate buffer, pH 7.4, for 24 h at 4°C. After rinsing with 0.2 M phosphate buffer (PBS), the specimens were fixed with 1% osmium oxide for 24 h at 4°C, dehydrated and freeze-dried with t-butyl alcohol (2-methyl-2-propanol). The dried specimens were coated with platinum, and the surface morphology was examined by SEM (JSM-6510, JEOL, Tokyo, Japan).
Isolation and cultivation of osteoblast-like cells
After being cut with an ROD or a UOD, the calvarial bone was extracted, and 8 8 mm bone blocks were further divided into two pieces. Primary osteoblast-like cells were isolated according to a protocol by Frosch et al. . The bone blocks were rinsed with 0.1 M PBS three times and placed on 35-mm cell culture dishes (BD Falcon, NJ, USA), covered with 0.5 ml culture medium and incubated at 37°C in a 100% humidified incubator containing an atmosphere of 95% air and 5% carbon dioxide. The culture medium was alpha MEM (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% foetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and streptomycin, penicillin and amphotericin B. After 30 minutes, another 1 ml of culture medium was added. After two weeks of culture, the cells were gently rinsed with 0.1 M PBS and incubated with 0.05% trypsin/EDTA (GIBCO, NY, USA). After being counted, the cells were resuspended and seeded in 60-mm culture dishes (BD Falcon, NJ, USA). After reaching confluence, cells were resuspended and seeded in 100-mm dishes (BD Falcon, NJ, USA) and used for the cell proliferation assay. In this experiment, 6 rats were used (3 in each group).
Cell proliferation assay
Cells were seeded into 96-well plates at 5000 cells/cm2, and cell counts were measured by colorimetry assay at 450 nm (Cell Counting Kit-8, Dojindo, Kumamoto, Japan) for 24, 48, 72, 120 (5 days) and 168 h (7 days). The medium was changed every 72 h. Experiments were performed in triplicate.
Cell differentiation assay (alkaline phosphatase and von Kossa staining)
Cells were re-seeded into 24-well plates at 5000 cells/cm2 in the culture medium. After 24 h, the same cells were stimulated with an osteoinduction medium consisting of 10% FBS, 50 μg/ml ascorbic acid, and 5 mM β-glycerophosphate. The medium was changed every 3 days. The cells were fixed and stained with alkaline phosphatase (ALP) stain at day 14 and von Kossa (VK) stain at day 28.
Statistical analyses were performed using JMP ver.14.0 software (SAS Institute Inc., NC, USA). One-way ANOVA was used for 3D and 2D bone morphometry. Student’s t-test was used to assess cell proliferation. p values of <0.05 indicated significant differences between the groups.