This study assesses the effect of low intensity pulsed ultrasound (LIPUS) on new bone formation during mandibular distraction osteogenesis (DO) in rabbits. 24 rabbits underwent DO on the right side of the mandible. 12 rabbits received a daily 20-min LIPUS (1.5 MHz, 30 mW/cm 2 ) treatment on the first day of the distraction until they were killed at week 0 (immediately after the distraction), week 2 and week 4 after the distraction. Four rabbits were killed at each time point. The other 12 rabbits followed the same protocol without the ultrasound treatment. A plain radiography, a micro-CT scan, a microhardness test and a histological examination were used to evaluate new bone formation in the distraction gap. At week 0 and week 2 after the distraction, the treatment groups showed higher radiopacity and microhardness ( p < 0.05), and more bone formation was detected by the histological examination. At week 4 after the distraction, there was no statistical difference between the two groups. In this study, LIPUS accelerated new bone formation during the distraction period and 2 weeks after the distraction, which implies that the effective time for using LIPUS is in the early stage of DO.
Distraction osteogenesis (DO) has the unique ability to gain new bone and simultaneously expand the surrounding soft tissues without a donor site. It has achieved worldwide acceptance and great success in the treatment of numerous congenital and acquired craniofacial skeletal anomalies . DO is a long term treatment, and it commonly takes 2–3 months for craniofacial DO . The rate-limiting step is the long waiting period for new bone formation, which takes at least two-thirds of the whole treatment time. Great interest has been focused on reducing the treatment time by accelerating new bone formation during DO.
Low intensity pulsed ultrasound (LIPUS) is a form of mechanical energy that is transmitted through living tissues as acoustic pressure waves. Many studies have found LIPUS to be effective in the acceleration of fracture healing . Two prospective, randomized, double-blind, placebo-controlled clinical trials have shown the same 38% reduction of healing time for tibial and radial fractures. The US Food and Drug Administration approved the use of LIPUS for the treatment of fresh fractures in 1994 and for the treatment of nonunion in 2000.
Several authors have reported the positive effects of LIPUS on long bone DO . Few studies have evaluated the effect of LIPUS on membranous bone DO. E l -B ialy et al. found that LIPUS accelerated new bone formation in a rabbit mandibular DO model, but S chortinghuis et al. showed that LIPUS did not appear to stimulate bone formation in the severely resorbed vertical distracted human mandible. More investigations are required to clarify the effect of LIPUS on membranous bone DO. The aim of the present study is to assess the effect of LIPUS on new bone formation during mandibular DO in rabbits.
Materials and methods
24 skeletally mature, male, New Zealand, white rabbits weighing 3.5–4.0 kg underwent DO on the right side of the mandible. In brief, anaesthesia was induced by intramuscular injections of ketamine (35 mg/kg) and diazepam (5 mg/kg). The skin on the right side of the mandible was shaved and disinfected with iodine solution, and the submandibular incision was 3 cm long. Once the mandibular body was exposed, the complete osteotomy was made just anterior to the first premolar straight down to the border of the mandible using a fissure bur. The custom-made distractor, a modification from an orthodontic palatal expansion screw (Hyrax, Germany) ( Fig. 1 ), was fixed to the mandible with 4 self-tapping titanium microscrews, and the distracting direction of the distractor was made parallel to the long axis of the mandibular body and perpendicular to the osteotomy line. The wounds were closed in layers. Postoperative care included wound care, and intramuscular injections of penicillin G sodium (0.5 million units) and acetaminophen (75 mg) each day for 3 days. After a 3-day latency period, the distraction was started at a rate of 0.5 mm/12 h for 10 days.
24 rabbits were randomly divided into two main groups: the ultrasound treatment group and the control (without ultrasound treatment) group ( n = 12). 4 rabbits from each group were killed at weeks 0, 2 and 4 after the distraction (week 0 means immediately after the distraction without a consolidation period).
A commercial ultrasound device was used. The Sonic Accelerated Fracture Healing System (Exogen Inc., Piscataway, NJ, USA) provided LIPUS with a 1.5 MHz frequency, modulated at 1 KHz with a signal burst width of 200 ms and an intensity of 30 mW/cm 2 . A single 20-min treatment to 12 rabbits per day started on the first day of the distraction and continued until they were killed at weeks 0, 2 and 4 after the distraction ( Fig. 1 ).
After the animal was euthanized with an intravenous injection of sodium pentobarbitone (1 ml/kg), its mandible was removed and separated at the synthesis, and a lateral film of the hemimandible (Kodak, Occlusal film, Ultra-speed, USA) was taken (10 mA, 50 KVP, 0.26 s, 12 in. FFD) with an aluminium step-wedge using an X-ray machine (Gendex, IL, USA) and processed by an automatic film processor (Dent X 9000, DentX/Logetronics GmbH, Germany). The films were transformed into digital images by a digital camera (JVC TK-C1380, Tokyo), and quantitative analysis was carried out with the software (Image Pro Plus 5.0, Media Cybernetics Inc., USA) to measure the mean grey level of the distraction gap that indirectly represents the projectional bone mineral density.
When the plain radiography was done, the specimen was cut from the distracted hemimandible including the distracted regeneration tissue and parts of the original bone, which was anterior and posterior to the distraction gap. Another horizontal cut along the long axis of the mandible body further bisected the specimen into upper and lower parts, and all the specimens were saline-soaked and frozen at −80 °C for preservation before additional testing.
The lower part of the specimen was scanned transversely with a section of 200 μm thickness (μCT20, Scano Medical AG, Switzerland). The bone volume fraction (%, bone volume/total volume) was calculated with software (Revision 3.1, Scanco Medical AG, Switzerland).
The upper part of the specimen underwent a microhardness using a Microhardness Tester (Buehler Micromed, England), and the Vickers microhardness (kg/mm 2 ), which represents the surface property of the distracted regeneration tissue, was used. The specimen was thawed to room temperature for at least 20 min before the test and the buccal surface of the distracted regeneration tissue was tested.
After the microhardness test, the upper part of the specimen was fixed in 10% formalin for 2 weeks, decalcified with 50% formic and 20% sodium citrate, then dehydrated in alcohol with increasing concentrations until it reached 100%, and finally embedded in paraffin. Sections of 5 μm thickness were cut longitudinally in the same direction as the distraction and stained with haematoxylin and eosin (H–E) for light microscopy examination (Carl Zeiss, Axioskop 40, Germany). In histomorphometric analysis, the distraction gap was longitudinally (the same as the direction of the distraction) divided into three horizontal rows (upper, middle and lower). In each horizontal row, four regions of interest (ROI) were chosen, two of which were near the original bone ends and the other two beside the middle of the distraction gap. 12 ROIs from one specimen were selected to represent the whole distraction gap. This sampling method was similar to that reported by C ope and S amchukov . The percentage of bone area (%, PBA) of each ROI was calculated with the software (Image Pro Plus 5.0, Media Cybernetics Inc., Slier Spring, USA). The PBA is the proportion of newly formed bone area to the total area. The software allows the researcher to select a specific colour to represent the new bone, so the total surface area of this same colour range is automatically calculated and the percentage determined. The PBA of each specimen was the mean total PBA of 12 ROIs.
All data were presented as the mean plus or minus the standard error of the mean (mean ± SEM). Differences between the treatment and control groups at each time point in mean grey level, bone volume fraction, microhardness and percentage of bone area were compared by the nonparametric Mann–Whitney test with SPSS (version 11, SPSS Inc., Chicago, IL, USA). The statistical significance was set at p < 0.05.
The animals tolerated the DO and ultrasound treatments well, since no infection or other complications occurred. The custom-made distractor showed excellent stability and strength, and no breakage or dislodgement of the distractors occurred.
In general, mineralization took place progressively from the ends of bone segments to the middle of the distraction gap decreasing the radiolucent zone in the middle of the distraction gap ( Fig. 2 ). At week 0 after the distraction, more bony spicules appeared in the distraction gap in the treatment group, and it had a significantly higher mean grey level (1.1492 ± 0.060) than the control group (0.9007 ± 0.054) ( p = 0.021).
At week 2 after the distraction, more radiopacity was noted in the treatment group. The mean grey level in the treatment group was 1.7637 ± 0.021, which was significantly higher than the 1.6502 ± 0.043 in the control group ( p = 0.043).
At week 4 after the distraction, the treatment group presented slightly more radiopacity than the control groups, but no significant difference was found ( p = 0.564). The mean grey level was 2.1181 ± 0.038 in the treatment group and 2.1091 ± 0.073 in the control group ( Fig. 3 ).
The image of each transverse section precisely showed the internal structure of the distracted regeneration tissue in the distraction gap. The difference in mineralization was visibly noticed between the two groups at week 0 after the distraction ( Fig. 4 ). The significant difference in bone volume fraction was found ( p = 0.021) to be 11 ± 2% in the treatment group and 3 ± 1% in the control group.
At week 2 after the distraction, dramatically increased mineralization and good continuity of the bony wall covering the distraction gap were seen, and slightly more radiopacity was noticed in the treatment group than in the control group. The difference in bone volume faction between the two groups was significant ( p = 0.043) with 42 ± 0.22% in the treatment group and 40 ± 1% in the control group.
At week 4 after the distraction, the two groups had similar radiological images. The bone volume faction showed no significant difference between them ( p = 0.564), but the treatment group had a higher value (53 ± 2%) than the control group (50 ± 3%).
At week 0 after the distraction, data were gained from three specimens in the treatment group and one specimen in the control group, the other specimens were too soft to be tested due to little bone formation on the surface of the distraction gap. Even though the data gained were too few to be of significance, three specimens in the treatment group had significantly higher microhardness than only one specimen in the control group ( Table 1 ).