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Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model
YIJOM-3353; No of Pages 7
Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx http://dx.doi.org/10.1016/j.ijom.2016.01.012, available online at http://www.sciencedirect.com
Research Paper Clinical Pathology
Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model
Z. Zhou1, M. Lang2, W. Fan3, X. Dong4, L. Zhu2, J. Xiao2, Y. Wang1 1
The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, China; 2School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, China; 3 Wenzhou Central Hospital, Wenzhou, China; 4 Wenzhou Integration Traditional Chinese and Western Medicine Hospital, Wenzhou, China
Z. Zhou, M. Lang, W. Fan, X. Dong, L. Zhu, J. Xiao, Y. Wang: Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model. Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx. # 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Abstract. 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. Microcomputed 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 0901-5027/000001+07
complications of irradiation and a notoriously refractory disease faced by oral and maxillofacial surgeons, seriously impairs the efficacy of radiotherapy. ORNJ is
Keywords: low-intensity ultrasound; osteoradionecrosis; radiotherapy; prevention; head and neck cancer; hyperbaric oxygen. Accepted for publication 25 January 2016
defined as an area that fails to heal over a period of 3–6 months in the absence of local neoplastic disease.1 There is currently no universally accepted treatment for
# 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhou Z, et al. Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.01.012
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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.2,3 Ultrasound is a safe, non-invasive, lowcost therapy, used routinely as a physical measure in the treatment of soft tissue disorders.4 Over the last decade, multiple studies have noted that low-intensity ultrasound effectively accelerates bone growth and bone fracture healing.5,6 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.7 Considering that low-intensity ultrasound can improve local blood circulation and stimulate collagen and bone production,8 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.8 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.9,10 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,1,11 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/ {a/b}], where n represents the number of irradiations, d the fractionated dose, and a/b approximates 3 in bone tissues), single 28-Gy doses are equivalent to the conventional fractionated doses of 80 Gy used in the clinical setting.12 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/cm2, a frequency of 1.5 MHz, a pulse breadth of 200 ms, and a pulse wave of 1 kHz frequency; this was repeated once daily, 20 min each time, for 30 days.7 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 mm 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 mm. The slices were baked at 60 8C 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 mm 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
Please cite this article in press as: Zhou Z, et al. Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.01.012
YIJOM-3353; No of Pages 7
Prevention of ORNJ by low-intensity US in the dog 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 x2 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). Results Physical surface observation
One week after radiation exposure, animals in groups A and B developed eating difficulties, salivation, dental mucosal ulcers, and other symptoms. Animals in group B showed little discomfort during ultrasound treatment. Three months after radiotherapy, unhealed tooth sockets, alveolar bone exposure, and the appearance of small sequestra in the mouth were generally observed in all animals in group A (Fig. 1A). However, 38 tooth sockets in 19 animals in group B were covered by new soft tissue, which was mildly hyperaemic, after 30 days of ultrasound treatment (Fig. 1B); only two tooth sockets in one animal had not healed. The
extraction wounds in the control group had healed. CBCT results
On CBCT, obvious alveolar bone resorption was observed around the root of the fourth premolar in group A animals, presenting with decreased bone density, thinning bone trabeculae, and an unsmooth, worm-eaten cortical bone surface due to irregular damage (Fig. 2A, D). However, there was no significant alveolar bone resorption in group B animals (Fig. 2B, E). Bone resorption was absent in control group animals (Fig. 2C, F). The alveolar bone density was 609.48 53.77 HU in group A, 829.65 81.46 HU in group B, and 1051.26 99.55 HU in the control group (P = 0.039). There were significant differences between groups: group A vs. group B, P = 0.008; group A vs. control group, P = 0.007; group B vs. control group, P = 0.013. The rate of occurrence of ORNJ
On the basis of the recovery of tooth sockets (as seen on physical surface observation) and alveolar bone resorption (as seen on CBCT), it was found that all animals in group A had developed ORNJ at 2 months after tooth extraction. In group B, the tooth sockets of 95% of the animals had tended to heal, but alveolar bone resorption was present in all animals, although it was less obvious than that in group A. Thus all animals in group B still developed ORNJ even with the application of ultrasound for 30 days. No animals in the control group developed ORNJ. These results indicate that low-intensity ultrasound is likely to fail to reduce the risk of occurrence of ORNJ.
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Micro-CT results
The various parameters of trabecular bone assessed in the three groups are shown in Table 1. The bone volume fraction, the ratio of bone surface area to bone volume, trabecular bone thickness, and the number of bone trabeculae in the control group were higher than those observed in groups A and B (P = 0.037, P = 0.033, P = 0.015, and P = 0.020, respectively); the values of these parameters were lower in group A than in group B (P = 0.037, P = 0.022, P = 0.017, and P = 0.034, respectively). The degree of separation and the trabecular pattern factor were higher in group A than in group B (P = 0.001 and P = 0.016, respectively), whereas those seen in the control group were lower than values in groups A and B (P = 0.019 and P = 0.008, respectively). HE staining results
In group A, the Haversian canals in many osteons were noted to have expanded significantly and there were several local huge lacunae; collagen fibres had lost their circular orientation (Fig. 3A). The osteons of group B tended to be normal, and the control group presented a typical cortical bone structure—a large number of osteons with visible alternately arranged annular layers of collagen fibres (Fig. 3B, C). CD34 immunofluorescence results
The expression of CD34 was weakly positive in group A, mildly positive in group B, and strongest in the control group (Fig. 4A–C), suggesting that almost no vascular tissue was present in group A and a small number of blood vessels were present in group B, but with a microvascular density still lower than that in the control group. Discussion
Fig. 1. Recovery of the tooth extraction wound at 2 months following mandibular tooth extraction (3 months after radiotherapy). (A) Experimental group A: the red arrow indicates where the wound at the tooth extraction site has not recovered and ORNJ has developed. (B) Experimental group B (30 days of low-intensity ultrasound treatment): the red arrow indicates where the wound at the tooth extraction site is covered by new, mildly hyperaemic soft tissue.
Although HBO has previously been regarded as an important preventive modality for ORNJ, there has been some debate on its effectiveness in the treatment of ORNJ. HBO is thought to improve the diffusion of oxygen in hypoxic tissues, which in turn stimulates collagen synthesis, matrix deposition, angiogenesis, and epithelialization. Marx et al. initially suggested the use of HBO before extraction for the prevention of ORNJ in irradiated tissue.13 According to this only randomized controlled trial, there were two cases (5.4%) of ORNJ in the HBO group, compared with 11 cases (29.9%) in the
Please cite this article in press as: Zhou Z, et al. Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.01.012
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Fig. 2. Coronal CBCT scans of mandibular fragments from the fourth premolar teeth: (A) experimental group A; (B) experimental group B (30 days of low-intensity ultrasound treatment); (C) control group. Sagittal CBCT scans of mandibular fragments from the fourth premolar teeth: (D) experimental group A; (E) experimental group B (30 days of low-intensity ultrasound treatment); (F) control group. There was obvious alveolar bone resorption in group A (A, D). In contrast, group B showed no significant alveolar bone resorption (B, E). The control group showed an absence of bone resorption (C, F).
prophylactic penicillin group. Inspired by this study, the investigators recommended prophylactic HBO to prevent ORNJ in post-radiation extraction patients. However, a randomized, placebo-controlled, double-blind trial from the ORN96 study, reported in 2004, questioned the effectiveness of HBO.3 That study failed to show any beneficial effect of HBO in patients with ORNJ and was stopped early. Some recent reviews have concluded that HBO should not be recommended in the routine management of patients with ORNJ.14,15 In the present study, a new prophylactic
treatment was assessed, namely low-intensity ultrasound, and it was attempted to confirm that this has a preventive effect on ORNJ. The single irradiation dose of 28 Gy has been demonstrated to be an ideal way to induce the formation of ORNJ.10 Therefore, this study used the same protocol to establish an animal model. The development of ORNJ was then determined on the basis of the recovery of tooth sockets combined with alveolar bone resorption, which has previously been confirmed to be a feasible measure for ORNJ.10 It was
found that all animals in group A presented unhealed tooth sockets and significant alveolar bone resorption, suggesting the successful establishment of radioactive mandibular injury in this dog model. Disappointingly, all animals in group B still developed ORNJ after ultrasound application for 30 days. However, some good signs were observed in group B animals, such as tooth sockets covered by new soft tissue and reduced alveolar bone resorption, which indicate that ultrasound treatment may improve the healing of ORNJ.
Please cite this article in press as: Zhou Z, et al. Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.01.012
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Prevention of ORNJ by low-intensity US in the dog
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Table 1. Micro-CT scanning results of bilateral mandibles in dogs.a Group A (Radiotherapy alone) Bone volume/total volume (%) Ratio of bone surface area to bone volume (1/mm) Thickness of trabecular bone (mm) Number of bone trabeculae (1/mm) Degree of separation of bone trabeculae (mm) Trabecular pattern factor (1/mm)
Group B (Radiotherapy followed by low-intensity ultrasound treatment)
Control group
P-value
0.1030 0.0143 5.5978 0.7307
0.1865 0.0290b,c 12.5360 2.5583b,c
0.2514 0.0467 19.0674 1.9561
0.037 0.033
0.0688 0.0128 0.5221 0.0635 1.3238 0.1961
0.1595 0.0391b,c 1.2266 0.1922b,c 0.7103 0.0867b
0.3572 0.0386 1.5760 0.3052 0.4749 0.1012
0.015 0.020 0.019
3.2275 0.3539
1.2245 0.3557b,c
0.7674 0.0921
0.008
a
Note: Data were calculated from 10 independent experiments and expressed as the mean standard deviation. Data were analyzed by oneway analysis of variance, followed by the least significant difference (LSD) post hoc test, which was used to calculate any difference between two groups. The P-value for the overall difference among the three groups was less than 0.05. b Statistically significant difference compared to group A (P < 0.05). c Statistically significant difference compared to the control group (P < 0.05).
It has been suggested that the irreversible damage to blood vessels caused by radiation plays a key role in the process of ORNJ development.16,17 Low-intensity ultrasound has been shown to promote angiogenesis in vivo18 and to have a direct vasodilator effect.19 Other studies have
reported that low-intensity ultrasound can stimulate mandibular cells, gingival fibroblasts, and peripheral blood mononuclear cells to produce a variety of angiogenic cytokines, such as interleukin 8, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor
(VEGF).20 This study found that after 30 days of ultrasound treatment, the microvascular density in group B dogs was significantly better than that in group A dogs, suggesting that in the process of ORNJ formation, ultrasound treatment may lead to a resistance to radiation
Fig. 3. Haematoxylin–eosin staining (200 magnification). (A) Experimental group A: Haversian canals in many osteons were seen to be significantly expanded (green arrows) and several local huge lacunae were observed; collagen fibres had lost their circular orientation (blue arrows). (B) Experimental group B: the osteons of group B tended to be normal. (C) Control group: the control group presented a large number of typical osteons, with visible alternately arranged annular layers of collagen fibres.
Fig. 4. Immunofluorescence staining using antibodies against CD34 (200 magnification). (A) Experimental group A; (B) experimental group B; (C) control group. The expression of CD34 was strongest in the control group. There was mild positive expression in group B and weak positive expression in group A. Positive sites are indicated by bright red fluorescence, compared with the relative darkness of negative areas.
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damage of blood vessels and an improvement in the blood supply to irradiated bone. Once blood flow to the injury site is increased, cell delivery and tissue oxygenation increase, thereby contributing to an early countering of the negative effects of hypocellularity, hypoxia, and hypovascularity.17 Likewise, a decrease in bone healing after irradiation is also an important factor with regard to ORNJ.21 Ultrasound can promote osteoblast and fibroblast proliferation and increase collagen and non-collagen protein synthesis, thereby enhancing bone formation and wound healing.20 The mechanism by which ultrasound promotes bone formation may be through upregulation of osteoprotegerin (OPG) and inhibition of receptor activator of nuclear factor kappa B ligand (RANKL) production in the bone microenvironment, thereby increasing bone regeneration.22 In addition, ultrasound stimulates the synthesis of nitric oxide and prostaglandin E2 in human osteoblasts, which are necessary for bone formation and remodelling.23 The results of this study showed that the bone density in group B was markedly better than that in group A. Micro-CT also indicated that the bone volume fraction, the ratio of bone surface area to bone volume, trabecular bone thickness, and the number of trabeculae in group B were greater than those in group A. Additionally, HE staining showed Haversian canals in osteons to be significantly expanded in group A, with collagen fibres losing their circular orientation, while group B tended to show typical osteons. Therefore, it is concluded that the osteogenic effect of low-intensity ultrasound may contribute to reducing the radiation toxicity to bone to some extent. The basis of low-intensity ultrasound to stimulate changes in tissues and cells has not been fully clarified. When ultrasound waves traverse through a tissue, vibrating forces are applied on every tissue component, such as intra- and extracellular fluids and cell membranes. These motions induce several physical effects, including thermal and non-thermal effects. Nonthermal mechanisms may largely be associated with the observed effects on the bone-healing process, since negligible temperature variations that are well below 1 8C have been reported after low-intensity ultrasound treatment.24,25 Non-thermal effects include stable cavitation, microstreaming, acoustic streaming, and direct mechanical effects on the cell membrane, which are able to induce a localized liquid flow as a result of pressure changes, facilitate the exchange of intra- and
extracellular ions and metabolites, alter cell membrane permeability to ions, and alter cell membrane electrophysiological properties, ultimately influencing cellular changes and responses.7 In conclusion, low-intensity ultrasound seems not to prevent the incidence of ORNJ in the dog. However, somewhat improved vascularity and bone quality at the microscopic level were found, which contributed to ORNJ healing. Further research is required to provide an in-depth understanding of this action on ORNJ. The intensity and frequency of the ultrasound signal used in this study were based on the optimal ultrasound parameters for promoting closed fracture healing. As the influences of ultrasound on bone depend greatly on the treatment regimen used, discussions of the optimal ultrasound setting should be included in all future research on the use of ultrasound for the prevention of ORNJ.
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Funding
10.
Funding was provided by the Medical Technology Project of the Department of Health in Zhejiang Province (2012KYA127).
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Competing interests
The authors declare that there are no conflicts of interest. 13.
Ethical approval
Animal care and all animal experiments were approved by the Animal Care and Use Committee of Wenzhou Medical University.
14.
Patient consent
15.
Not required. References 1. Nabil S, Samman N. Incidence and prevention of osteoradionecrosis after dental extraction in irradiated patients: a systematic review. Int J Oral Maxillofac Surg 2011;40: 229–43. 2. Chuang S. Limited evidence to demonstrate that the use of hyperbaric oxygen (HBO) therapy reduces the incidence of osteoradionecrosis in irradiated patients requiring tooth extraction. J Evid Based Dent Pract 2012;12:248–50. 3. Annane D, Depondt J, Aubert P, Villart M, Ge´hanno P, Gajdos P, et al. Hyperbaric oxygen therapy for radionecrosis of the jaw: a randomized, placebo-controlled, double-blind
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trial from the ORN96 study group. J Clin Oncol 2004;22:4893–900. Speed C. Therapeutic ultrasound in soft tissue lesions. Rheumatology 2001;40: 1331–6. Claes L, Willie B. The enhancement of bone regeneration by ultrasound. Prog Biophys Mol Biol 2007;93:384–98. Pounder NM, Harrison AJ. Low intensity pulsed ultrasound for fracture healing: a review of the clinical evidence and the associated biological mechanism of action. Ultrasonics 2008;48:330–8. Schortinghuis J, Stegenga B, Raghoebar G, de Bont L. Ultrasound stimulation of maxillofacial bone healing. Crit Rev Oral Biol Med 2003;14:63–74. Harris M. The conservative management of osteoradionecrosis of the mandible with ultrasound therapy. Br J Oral Maxillofac Surg 1992;30:313–8. Wu G, Chen L, Zhu G, Wang Y. Low-intensity ultrasound accelerates mandibular implant bone integration in dogs with mandibular osteoradionecrosis. J Surg Res 2013;182:55–61. Wu G, Chen L, Qu T, Zhu G, Wang Y, Zhu C. Ultrasonic treatment of canine ORNM. J Maxillofac Oral Surg 2013;71:199–207. Reher P, Harris M. Ultrasound for the treatment of osteoradionecrosis. J Maxillofac Oral Surg 1997;55:1193–4. Cohen M, Nishimura I, Tamplen M, Hokugo A, Beumer J, Steinberg ML, et al. Animal model of radiogenic bone damage to study mandibular osteoradionecrosis. Am J Otolaryngol 2011;32:291–300. Marx RE, Johnson RP, Kline SN. Prevention of osteoradionecrosis: a randomized prospective clinical trial of hyperbaric oxygen versus penicillin. J Am Dent Assoc 1985;111: 49–54. Bessereau J, Annane D. Treatment of osteoradionecrosis of the jaw: the case against the use of hyperbaric oxygen. J Maxillofac Oral Surg 2010;68:1907–10. Shaw RJ, Butterworth C. Hyperbaric oxygen in the management of late radiation injury to the head and neck. Part II: prevention. Br J Oral Maxillofac Surg 2011;49:9–13. Marx RE, Johnson RP. Studies in the radiobiology of osteoradionecrosis and their clinical significance. Oral Surg Oral Med Oral Pathol 1987;64:379–90. Marx RE. A new concept in the treatment of osteoradionecrosis. J Maxillofac Oral Surg 1983;41:351–7. Ramli R, Reher P, Harris M, Meghji S. The effect of ultrasound on angiogenesis: an in vivo study using the chick chorioallantoic membrane. Int J Oral Maxillofac Implants 2009;24:591–6. Rawool NM, Goldberg BB, Forsberg F, Winder AA, Hume E. Power Doppler assessment of vascular changes during fracture treatment with low-intensity ultrasound. J Ultrasound Med 2003;22:145–53.
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RANKL ratio in human osteoblast-like cells. Bone 2006;39:283–8. 23. Reher P, Harris M, Whiteman M, Hai H, Meghji S. Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts. Bone 2002;31:236–41. 24. Chang WH, Sun JS, Chang SP, Lin JC. Study of thermal effects of ultrasound stimulation on fracture healing. Bioelectromagnetics 2002;23:256–63. 25. Duarte L. The stimulation of bone growth by ultrasound. Arch Orthop Trauma Surg 1983;101:153–9.
Address: Yanliang Wang Wenzhou Medical University The Second Affiliated Hospital No. 109 West Xueyuan Road Wenzhou Zhejiang 325027 China Tel.: +86 577 88066010; Fax: +86 577 88066081 E-mail: [email protected]
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Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx http://dx.doi.org/10.1016/j.ijom.2016.01.012, available online at http://www.sciencedirect.com
Research Paper Clinical Pathology
Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model
Z. Zhou1, M. Lang2, W. Fan3, X. Dong4, L. Zhu2, J. Xiao2, Y. Wang1 1
The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, China; 2School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, China; 3 Wenzhou Central Hospital, Wenzhou, China; 4 Wenzhou Integration Traditional Chinese and Western Medicine Hospital, Wenzhou, China
Z. Zhou, M. Lang, W. Fan, X. Dong, L. Zhu, J. Xiao, Y. Wang: Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model. Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx. # 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Abstract. 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. Microcomputed 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 0901-5027/000001+07
complications of irradiation and a notoriously refractory disease faced by oral and maxillofacial surgeons, seriously impairs the efficacy of radiotherapy. ORNJ is
Keywords: low-intensity ultrasound; osteoradionecrosis; radiotherapy; prevention; head and neck cancer; hyperbaric oxygen. Accepted for publication 25 January 2016
defined as an area that fails to heal over a period of 3–6 months in the absence of local neoplastic disease.1 There is currently no universally accepted treatment for
# 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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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.2,3 Ultrasound is a safe, non-invasive, lowcost therapy, used routinely as a physical measure in the treatment of soft tissue disorders.4 Over the last decade, multiple studies have noted that low-intensity ultrasound effectively accelerates bone growth and bone fracture healing.5,6 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.7 Considering that low-intensity ultrasound can improve local blood circulation and stimulate collagen and bone production,8 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.8 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.9,10 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,1,11 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/ {a/b}], where n represents the number of irradiations, d the fractionated dose, and a/b approximates 3 in bone tissues), single 28-Gy doses are equivalent to the conventional fractionated doses of 80 Gy used in the clinical setting.12 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/cm2, a frequency of 1.5 MHz, a pulse breadth of 200 ms, and a pulse wave of 1 kHz frequency; this was repeated once daily, 20 min each time, for 30 days.7 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 mm 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 mm. The slices were baked at 60 8C 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 mm 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
Please cite this article in press as: Zhou Z, et al. Prevention of osteoradionecrosis of the jaws by low-intensity ultrasound in the dog model, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.01.012
YIJOM-3353; No of Pages 7
Prevention of ORNJ by low-intensity US in the dog 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 x2 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). Results Physical surface observation
One week after radiation exposure, animals in groups A and B developed eating difficulties, salivation, dental mucosal ulcers, and other symptoms. Animals in group B showed little discomfort during ultrasound treatment. Three months after radiotherapy, unhealed tooth sockets, alveolar bone exposure, and the appearance of small sequestra in the mouth were generally observed in all animals in group A (Fig. 1A). However, 38 tooth sockets in 19 animals in group B were covered by new soft tissue, which was mildly hyperaemic, after 30 days of ultrasound treatment (Fig. 1B); only two tooth sockets in one animal had not healed. The
extraction wounds in the control group had healed. CBCT results
On CBCT, obvious alveolar bone resorption was observed around the root of the fourth premolar in group A animals, presenting with decreased bone density, thinning bone trabeculae, and an unsmooth, worm-eaten cortical bone surface due to irregular damage (Fig. 2A, D). However, there was no significant alveolar bone resorption in group B animals (Fig. 2B, E). Bone resorption was absent in control group animals (Fig. 2C, F). The alveolar bone density was 609.48 53.77 HU in group A, 829.65 81.46 HU in group B, and 1051.26 99.55 HU in the control group (P = 0.039). There were significant differences between groups: group A vs. group B, P = 0.008; group A vs. control group, P = 0.007; group B vs. control group, P = 0.013. The rate of occurrence of ORNJ
On the basis of the recovery of tooth sockets (as seen on physical surface observation) and alveolar bone resorption (as seen on CBCT), it was found that all animals in group A had developed ORNJ at 2 months after tooth extraction. In group B, the tooth sockets of 95% of the animals had tended to heal, but alveolar bone resorption was present in all animals, although it was less obvious than that in group A. Thus all animals in group B still developed ORNJ even with the application of ultrasound for 30 days. No animals in the control group developed ORNJ. These results indicate that low-intensity ultrasound is likely to fail to reduce the risk of occurrence of ORNJ.
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Micro-CT results
The various parameters of trabecular bone assessed in the three groups are shown in Table 1. The bone volume fraction, the ratio of bone surface area to bone volume, trabecular bone thickness, and the number of bone trabeculae in the control group were higher than those observed in groups A and B (P = 0.037, P = 0.033, P = 0.015, and P = 0.020, respectively); the values of these parameters were lower in group A than in group B (P = 0.037, P = 0.022, P = 0.017, and P = 0.034, respectively). The degree of separation and the trabecular pattern factor were higher in group A than in group B (P = 0.001 and P = 0.016, respectively), whereas those seen in the control group were lower than values in groups A and B (P = 0.019 and P = 0.008, respectively). HE staining results
In group A, the Haversian canals in many osteons were noted to have expanded significantly and there were several local huge lacunae; collagen fibres had lost their circular orientation (Fig. 3A). The osteons of group B tended to be normal, and the control group presented a typical cortical bone structure—a large number of osteons with visible alternately arranged annular layers of collagen fibres (Fig. 3B, C). CD34 immunofluorescence results
The expression of CD34 was weakly positive in group A, mildly positive in group B, and strongest in the control group (Fig. 4A–C), suggesting that almost no vascular tissue was present in group A and a small number of blood vessels were present in group B, but with a microvascular density still lower than that in the control group. Discussion
Fig. 1. Recovery of the tooth extraction wound at 2 months following mandibular tooth extraction (3 months after radiotherapy). (A) Experimental group A: the red arrow indicates where the wound at the tooth extraction site has not recovered and ORNJ has developed. (B) Experimental group B (30 days of low-intensity ultrasound treatment): the red arrow indicates where the wound at the tooth extraction site is covered by new, mildly hyperaemic soft tissue.
Although HBO has previously been regarded as an important preventive modality for ORNJ, there has been some debate on its effectiveness in the treatment of ORNJ. HBO is thought to improve the diffusion of oxygen in hypoxic tissues, which in turn stimulates collagen synthesis, matrix deposition, angiogenesis, and epithelialization. Marx et al. initially suggested the use of HBO before extraction for the prevention of ORNJ in irradiated tissue.13 According to this only randomized controlled trial, there were two cases (5.4%) of ORNJ in the HBO group, compared with 11 cases (29.9%) in the
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Fig. 2. Coronal CBCT scans of mandibular fragments from the fourth premolar teeth: (A) experimental group A; (B) experimental group B (30 days of low-intensity ultrasound treatment); (C) control group. Sagittal CBCT scans of mandibular fragments from the fourth premolar teeth: (D) experimental group A; (E) experimental group B (30 days of low-intensity ultrasound treatment); (F) control group. There was obvious alveolar bone resorption in group A (A, D). In contrast, group B showed no significant alveolar bone resorption (B, E). The control group showed an absence of bone resorption (C, F).
prophylactic penicillin group. Inspired by this study, the investigators recommended prophylactic HBO to prevent ORNJ in post-radiation extraction patients. However, a randomized, placebo-controlled, double-blind trial from the ORN96 study, reported in 2004, questioned the effectiveness of HBO.3 That study failed to show any beneficial effect of HBO in patients with ORNJ and was stopped early. Some recent reviews have concluded that HBO should not be recommended in the routine management of patients with ORNJ.14,15 In the present study, a new prophylactic
treatment was assessed, namely low-intensity ultrasound, and it was attempted to confirm that this has a preventive effect on ORNJ. The single irradiation dose of 28 Gy has been demonstrated to be an ideal way to induce the formation of ORNJ.10 Therefore, this study used the same protocol to establish an animal model. The development of ORNJ was then determined on the basis of the recovery of tooth sockets combined with alveolar bone resorption, which has previously been confirmed to be a feasible measure for ORNJ.10 It was
found that all animals in group A presented unhealed tooth sockets and significant alveolar bone resorption, suggesting the successful establishment of radioactive mandibular injury in this dog model. Disappointingly, all animals in group B still developed ORNJ after ultrasound application for 30 days. However, some good signs were observed in group B animals, such as tooth sockets covered by new soft tissue and reduced alveolar bone resorption, which indicate that ultrasound treatment may improve the healing of ORNJ.
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Prevention of ORNJ by low-intensity US in the dog
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Table 1. Micro-CT scanning results of bilateral mandibles in dogs.a Group A (Radiotherapy alone) Bone volume/total volume (%) Ratio of bone surface area to bone volume (1/mm) Thickness of trabecular bone (mm) Number of bone trabeculae (1/mm) Degree of separation of bone trabeculae (mm) Trabecular pattern factor (1/mm)
Group B (Radiotherapy followed by low-intensity ultrasound treatment)
Control group
P-value
0.1030 0.0143 5.5978 0.7307
0.1865 0.0290b,c 12.5360 2.5583b,c
0.2514 0.0467 19.0674 1.9561
0.037 0.033
0.0688 0.0128 0.5221 0.0635 1.3238 0.1961
0.1595 0.0391b,c 1.2266 0.1922b,c 0.7103 0.0867b
0.3572 0.0386 1.5760 0.3052 0.4749 0.1012
0.015 0.020 0.019
3.2275 0.3539
1.2245 0.3557b,c
0.7674 0.0921
0.008
a
Note: Data were calculated from 10 independent experiments and expressed as the mean standard deviation. Data were analyzed by oneway analysis of variance, followed by the least significant difference (LSD) post hoc test, which was used to calculate any difference between two groups. The P-value for the overall difference among the three groups was less than 0.05. b Statistically significant difference compared to group A (P < 0.05). c Statistically significant difference compared to the control group (P < 0.05).
It has been suggested that the irreversible damage to blood vessels caused by radiation plays a key role in the process of ORNJ development.16,17 Low-intensity ultrasound has been shown to promote angiogenesis in vivo18 and to have a direct vasodilator effect.19 Other studies have
reported that low-intensity ultrasound can stimulate mandibular cells, gingival fibroblasts, and peripheral blood mononuclear cells to produce a variety of angiogenic cytokines, such as interleukin 8, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor
(VEGF).20 This study found that after 30 days of ultrasound treatment, the microvascular density in group B dogs was significantly better than that in group A dogs, suggesting that in the process of ORNJ formation, ultrasound treatment may lead to a resistance to radiation
Fig. 3. Haematoxylin–eosin staining (200 magnification). (A) Experimental group A: Haversian canals in many osteons were seen to be significantly expanded (green arrows) and several local huge lacunae were observed; collagen fibres had lost their circular orientation (blue arrows). (B) Experimental group B: the osteons of group B tended to be normal. (C) Control group: the control group presented a large number of typical osteons, with visible alternately arranged annular layers of collagen fibres.
Fig. 4. Immunofluorescence staining using antibodies against CD34 (200 magnification). (A) Experimental group A; (B) experimental group B; (C) control group. The expression of CD34 was strongest in the control group. There was mild positive expression in group B and weak positive expression in group A. Positive sites are indicated by bright red fluorescence, compared with the relative darkness of negative areas.
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damage of blood vessels and an improvement in the blood supply to irradiated bone. Once blood flow to the injury site is increased, cell delivery and tissue oxygenation increase, thereby contributing to an early countering of the negative effects of hypocellularity, hypoxia, and hypovascularity.17 Likewise, a decrease in bone healing after irradiation is also an important factor with regard to ORNJ.21 Ultrasound can promote osteoblast and fibroblast proliferation and increase collagen and non-collagen protein synthesis, thereby enhancing bone formation and wound healing.20 The mechanism by which ultrasound promotes bone formation may be through upregulation of osteoprotegerin (OPG) and inhibition of receptor activator of nuclear factor kappa B ligand (RANKL) production in the bone microenvironment, thereby increasing bone regeneration.22 In addition, ultrasound stimulates the synthesis of nitric oxide and prostaglandin E2 in human osteoblasts, which are necessary for bone formation and remodelling.23 The results of this study showed that the bone density in group B was markedly better than that in group A. Micro-CT also indicated that the bone volume fraction, the ratio of bone surface area to bone volume, trabecular bone thickness, and the number of trabeculae in group B were greater than those in group A. Additionally, HE staining showed Haversian canals in osteons to be significantly expanded in group A, with collagen fibres losing their circular orientation, while group B tended to show typical osteons. Therefore, it is concluded that the osteogenic effect of low-intensity ultrasound may contribute to reducing the radiation toxicity to bone to some extent. The basis of low-intensity ultrasound to stimulate changes in tissues and cells has not been fully clarified. When ultrasound waves traverse through a tissue, vibrating forces are applied on every tissue component, such as intra- and extracellular fluids and cell membranes. These motions induce several physical effects, including thermal and non-thermal effects. Nonthermal mechanisms may largely be associated with the observed effects on the bone-healing process, since negligible temperature variations that are well below 1 8C have been reported after low-intensity ultrasound treatment.24,25 Non-thermal effects include stable cavitation, microstreaming, acoustic streaming, and direct mechanical effects on the cell membrane, which are able to induce a localized liquid flow as a result of pressure changes, facilitate the exchange of intra- and
extracellular ions and metabolites, alter cell membrane permeability to ions, and alter cell membrane electrophysiological properties, ultimately influencing cellular changes and responses.7 In conclusion, low-intensity ultrasound seems not to prevent the incidence of ORNJ in the dog. However, somewhat improved vascularity and bone quality at the microscopic level were found, which contributed to ORNJ healing. Further research is required to provide an in-depth understanding of this action on ORNJ. The intensity and frequency of the ultrasound signal used in this study were based on the optimal ultrasound parameters for promoting closed fracture healing. As the influences of ultrasound on bone depend greatly on the treatment regimen used, discussions of the optimal ultrasound setting should be included in all future research on the use of ultrasound for the prevention of ORNJ.
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Funding
10.
Funding was provided by the Medical Technology Project of the Department of Health in Zhejiang Province (2012KYA127).
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Competing interests
The authors declare that there are no conflicts of interest. 13.
Ethical approval
Animal care and all animal experiments were approved by the Animal Care and Use Committee of Wenzhou Medical University.
14.
Patient consent
15.
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RANKL ratio in human osteoblast-like cells. Bone 2006;39:283–8. 23. Reher P, Harris M, Whiteman M, Hai H, Meghji S. Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts. Bone 2002;31:236–41. 24. Chang WH, Sun JS, Chang SP, Lin JC. Study of thermal effects of ultrasound stimulation on fracture healing. Bioelectromagnetics 2002;23:256–63. 25. Duarte L. The stimulation of bone growth by ultrasound. Arch Orthop Trauma Surg 1983;101:153–9.
Address: Yanliang Wang Wenzhou Medical University The Second Affiliated Hospital No. 109 West Xueyuan Road Wenzhou Zhejiang 325027 China Tel.: +86 577 88066010; Fax: +86 577 88066081 E-mail: [email protected]
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