Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model


The prevention of osteoradionecrosis of the jaws (ORNJ) is very important because of the current absence of effective therapies for this disease. The aim of this study was to determine whether low-intensity ultrasound has a preventive effect on ORNJ. Sixty healthy adult dogs were divided randomly into three groups: group A (radiotherapy alone), group B (radiotherapy followed by low-intensity ultrasound treatment), and a control group. The development of ORNJ was assessed and the rate of occurrence of ORNJ was compared between groups A and B. Micro-computed tomography, haematoxylin–eosin staining, and immunofluorescence were used to evaluate the microstructure of the mandible and changes in microvascular density in all groups. All animals in group A and group B (ultrasound applied for 30 days) developed ORNJ. Alveolar bone density was 609.48 ± 53.77 HU in group A and 829.65 ± 81.46 HU in group B ( P = 0.008). The trabecular bone volume fraction, bone surface area/bone volume ratio, trabecular thickness, and trabecular number were all lower in group A than in group B ( P = 0.037, P = 0.022, P = 0.017, and P = 0.034, respectively). Haematoxylin–eosin staining showed that the Haversian canals in the osteons had expanded significantly in group A, with collagen fibres losing their circular orientation; group B tended to show typical osteons. The microvascular density in group A was decreased. In conclusion, the use of low-intensity ultrasound in the dog appears not to prevent the incidence of ORNJ, however it does somewhat improve vascularity and bone quality at the microscopic level, which contribute to ORNJ healing.

Radiotherapy is used widely in the treatment of head and neck cancer following radical surgery. However, osteoradionecrosis of the jaws (ORNJ), one of the worst complications of irradiation and a notoriously refractory disease faced by oral and maxillofacial surgeons, seriously impairs the efficacy of radiotherapy. ORNJ is defined as an area that fails to heal over a period of 3–6 months in the absence of local neoplastic disease. There is currently no universally accepted treatment for this chronic pathological condition and therefore the prevention of ORNJ after radiotherapy has become a widespread concern. Hyperbaric oxygen (HBO) has been applied to patients before and after radiotherapy in an attempt to reduce the risk of ORNJ occurring and is a popular preventive measure. However, the main drawbacks of HBO, including controversies surrounding its efficacy, the high price, and its low availability, have greatly limited its clinical application.

Ultrasound is a safe, non-invasive, low-cost therapy, used routinely as a physical measure in the treatment of soft tissue disorders. Over the last decade, multiple studies have noted that low-intensity ultrasound effectively accelerates bone growth and bone fracture healing. The acceleration of fracture repair seems to be multilevel, involving different cell types, such as fibroblasts, osteoprogenitor cells, and chondroblasts, and different steps in the fracture-healing process, including the inflammatory, reparative, and remodelling phases. Considering that low-intensity ultrasound can improve local blood circulation and stimulate collagen and bone production, could it also promote the healing of blood vessels and bone tissues injured by radiotherapy? Harris was the first author to use therapeutic ultrasound for the treatment of mandibular osteoradionecrosis. In that study it was found that 10/21 (48%) cases achieved healing after treatment with debridement and ultrasound alone, without the need for surgery or sophisticated technology. Nine further patients were treated successfully by covering the preserved mandible with a local intraoral flap. Wu et al. recently established a dog model of ORNJ to demonstrate that low-intensity ultrasound can improve the healing of irradiated bone. After ultrasound treatment, microvessel density and the microarchitecture of the mandible, as well as metabolism in osteoblasts, increased significantly.

Some researchers have proposed the use of ultrasound for the prevention of ORNJ in patients after radiotherapy and as an alternative to HBO, as it is economical, readily available, and simple to perform. Since few relevant in vivo studies have been reported in the literature, this study was performed to explore the preventive effect of low-intensity ultrasound on the development of ORNJ after radiotherapy and to further study the mechanisms of its potential prophylactic effect, with the aim of developing a theoretical basis for its clinical application as a new preventive measure.

Materials and methods

Establishment of the animal model

All procedures on animals were done in accordance with the guidelines of the animal care and use committee of the study institution. A total of 60 healthy adult female and male crossbred dogs (2 to 3 years old; weight 12 to 17 kg) were selected for this study. The dogs were divided randomly into experimental group A, experimental group B, and a control group, with 20 in each group. The animals in the two experimental groups underwent radiotherapy, and those in the control group did not.

Animals were anaesthetized by injection of a 35-mg/kg dose of 3% pentobarbital sodium into the great saphenous vein. Radiation was administered using a Varian 600 C/D electron accelerator (Varian Medical Systems, Palo Alto, CA, USA); three-dimensional (3D) stereotactic radiotherapy was applied on the bilateral mandibular posterior teeth. The remaining locations were covered by a lead plate. Single doses of radiotherapy were given at 28 Gy using 1-cm step stereotactic radiotherapy. According to the biological equivalent dose linear quadratic equation (biological equivalent dose = nd [1 + d /{ α / β }], where n represents the number of irradiations, d the fractionated dose, and α/β approximates 3 in bone tissues), single 28-Gy doses are equivalent to the conventional fractionated doses of 80 Gy used in the clinical setting. A liquid diet was administered to the dogs with radiation-induced injuries, such as dental ulcers, at an early stage.

Low-intensity ultrasound treatment

After radiotherapy, animals in group B were treated immediately with low-intensity ultrasound using a US10 ultrasound therapeutic apparatus (Xingwan Electronic Instrument, Hong Kong, China); animals in group A and the control group did not receive ultrasound treatment. The ultrasound was applied at an intensity of 30 mW/cm 2 , a frequency of 1.5 MHz, a pulse breadth of 200 μs, and a pulse wave of 1 kHz frequency; this was repeated once daily, 20 min each time, for 30 days. The area of the mandibular teeth was chosen.

One month after radiotherapy, the bilateral mandibular fourth premolars were extracted from all animals in the three groups under aseptic conditions. The preventive effect of ultrasound was evaluated using cone beam computed tomography (CBCT) (Quantitative Radiology, Verona, Italy), and the images captured were reconstructed using Simplant software (Materialise, Leuven, Belgium). Alveolar bone density was measured and recorded in Hounsfield units (HU).

Rate of occurrence of ORNJ

Two months after tooth extraction, the animals were anaesthetized as described above. The occurrence of ORNJ was determined by assessment of the recovery of the tooth socket in combination with the alveolar bone resorption observed on CBCT scanning. ORNJ was deemed to have occurred when the tooth socket had not healed and there was alveolar bone resorption. The rate of occurrence of ORNJ was compared between groups A and B.

Micro-computed tomography (micro-CT) analysis

Mandibular peri-premolar tissues were extracted. Samples measuring 5 × 5 × 5 mm were prepared from small sections and these were placed under a CT microscope. The Inveon micro-CT system (Siemens, Munich, Germany) was used to perform sample scanning. Cubes with a side length of 300 μm were selected on the images (grey values of 67–127). The images were then subjected to 3D analysis using the micro-CT system internal software. Various spatial indices of trabecular bone were measured.

Haematoxylin–eosin (HE) staining

After micro-CT scanning, the mandibles were fixed in 20% paraformaldehyde solution for 2 days. Partial specimens were subjected to ethanol gradient dehydration, treated with dimethylbenzene hyaline, and embedded in organic glass. Consecutive slicing was performed using a heavy hard texture slicer at a slice thickness of 5 μm. The slices were baked at 60 °C for 5 days and then subjected to HE staining. At this point, the samples were observed under a DMLA automated microscope (Leica, Solms, Germany).

CD34 immunofluorescence

The remaining bone pieces were demineralized with decalcification liquid and embedded in paraffin. Consecutive slices of 5 μm in thickness were obtained and subjected to conventional slice baking. CD34 antibody was detected using the streptavidin–biotin–peroxidase complex method (Beijing Biosynthesis Biotechnology, Beijing, China) and immunofluorescence staining. Changes in microvessel intensity were observed under a fluorescence microscope. Positive expression of CD34 was represented by red cytoplasm in the vascular endothelial cells.

Statistical analysis

Data were expressed as mean ± standard deviation (SD) values. The rate of occurrence of ORNJ in group A was compared with that in group B by χ 2 test. The results obtained from CBCT and micro-CT scanning were assessed, and statistical differences among the groups were analyzed by one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) post hoc test, which was used to calculate any differences between two groups. A P -value of <0.05 was considered statistically significant. All analyses were done using SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA).

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Dec 15, 2017 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model
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