Device description
A NiTi mesh device was used (Ni, 56.1 wt%; Ti balance; Fe, 0.05 wt%; O, 0.05 wt%; C, 0.03 wt%; N, 0.02 wt%). The mesh device was 5 mm in width, 25 mm in length, and 0.275 mm in thickness. The mesh was curved such that the middle point was 4 mm above the “baseline”.
The deformation behavior of the SMA mesh plate was assessed by compressive testing. The plate was fixed to an Instron testing machine (Model AG-I; Shimadzu, Kyoto, Japan) and compressive stress was applied perpendicularly at a crosshead speed of 1.0 mm min−1. Compressive load and displacement were calculated when the plate had completely flattened.
The center region and entire plate were 5 and 15 mm in width, respectively. The compressive load required for identical displacement of the two regions is 2.7 N (= 0.9 ÷ 5 × 15). This is lower than the measured compressive load applied to the whole plate, likely due to the porosity of the center of the plate.
Surgical protocol
Male Japanese white rabbits (3–3.5 kg) were divided into four groups of three, according to the time point of euthanasia and collagen membrane coverage status. The protocol and guidelines for this study were reviewed and approved by the Animal Care and Use Committee of Tohoku University, Sendai, Japan (2018SHIDO-030).
The animals were anaesthetized by intramuscular administration of ketamine hydrochloride (60 mg/kg Ketalar; Sankyo, Tokyo, Japan), followed by diazepam (5 mg) and atropine sulphate (0.5 mg), without endotracheal intubation. Before the operation, 10 mg/kg pentobarbital sodium was injected intravenously. In addition, 1.8 mL of local anesthetic (2% xylocaine and epinephrine 1:80,000; Dentsply Sankin, Tokyo, Japan) were used during all surgical procedures.
The operation was performed under standard sterile conditions. The forehead of the animal was shaved and disinfected with 1% iodine sodium. After U-shaped skin and periosteal incisions were made in the forehead region, the frontal bone of the animal was exposed following careful elevation of the periosteum. A titanium screw (1.4 mm in diameter, 3 mm in length; Jeil Medical, Seoul, Korea) was inserted into the bone surface to anchor the SMA device using resorbable threads. The device was attached to the bony surface and fixed with 5–0 Vicryl Rapide® resorbable thread (Johnson & Johnson, New Brunswick, NJ, USA). The cortical bone was perforated by drilling through the SMA mesh using a round bur. In half of the rabbits in the experimental group, the SMA device was covered with a resorbable atelocollagen membrane (Koken Tissue Guide; Olympus Terumo Biomaterials, Tokyo, Japan). After irrigation with saline, the periosteum was replaced and stabilized using 5–0 Vicryl (Johnson & Johnson) sutures (Fig. 1). The skin was closed using 4–0 Vicryl sutures. Animals received cefazolin sodium (20 mg/kg) subcutaneously until postoperative day 3.
The rabbits were provided with water and a commercial rabbit diet postoperatively. Rabbits were euthanized with a lethal dose of thiopental sodium at 5 or 8 weeks after surgery. The cranial bone was removed and fixed for 14 days in 10% buffered formalin. The rabbits were divided into groups C1 (5 weeks postoperatively without membrane; n = 3), C2 (8 weeks postoperatively without membrane; n = 3), E1 (5 weeks postoperatively with membrane; n = 3), and E2 (8 weeks postoperatively with membrane; n = 3).
Tissue preparation and histological evaluation
Cranial bone tissue was evaluated by X-ray computed tomography (CT) (Comscantecno, Co., Ltd., Yokohama, Japan) operating at 65 µA and 70 kV. Measurements were made on three vertical images per specimen, as close as possible to the center of the device. In each image, the area occupied by new bone was measured using ImageJ software (ver. 1.44; NIH, Bethesda, MD, USA). The area beneath the SMA device was defined as the expanded volume (EV). The area of mineralized tissue in the EV was defined as the total bone volume (TBV) (Fig. 2). The EV/TBV ratio was calculated to assess the extent of new bone growth.
After micro-CT, specimens were decalcified in phosphate-buffered saline (PBS) with 10% ethylenediaminetetraacetic acid (EDTA) at room temperature for 60 days. The specimens were then dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Sagittal sections of 5 µm thickness were made using a microtome and mounted on glass slides. Hematoxylin and eosin and elastica and Masson staining were performed on serial sections for morphological evaluation of newly formed bone in the gap between the SMA device and surface of the original cranial bone.
Statistical analysis
Normality and homogeneity were analyzed first, and an unpaired Student’s t-test was used to evaluate the area of newly formed bone and the EV/TBV ratio. The level of significance was set at P < 0.05.