Abstract
Objective
The objective of the study was to create an animal model to study mandibular osteoradionecrosis (ORN) using high–dose rate (HDR) brachytherapy.
Methods
Ten Sprague-Dawley male rats were used in this study. Six rats received a single dose of 30 Gy using an HDR remote afterloading machine via a brachytherapy catheter placed along the left hemimandible. The remaining 4 rats served as controls with catheter placement without radiation (sham). On the day following irradiation or sham, all 3 left mandibular molars were atraumatically extracted. Twenty-eight days after irradiation, mandibles were examined using nondecalcified histology with sequential fluorochrome labeling, decalcified histology, and micro–computed tomography scanning.
Results
Irradiated rats demonstrated exposed bone at the extraction sockets, whereas the control animals had complete mucosalization. Alopecia was also seen in the irradiated group. Both histologic and radiologic analyses of the mandible specimens demonstrated a reduction in bone formation in the radiated mandibles as compared with controls.
Conclusions
Our HDR brachytherapy model incorporating postradiation dental extractions has successfully demonstrated reproducible radiogenic mandibular bone damage analogous to the clinical ORN. Although clinical criteria continue to be used today in describing ORN, this model can serve as a platform for future studies to define ORN and delineate its pathogenesis.
1
Introduction
Despite more target-specific advances in radiation technology for the treatment of head and neck cancer, including intensity-modulated radiation therapy and high–dose rate (HDR) brachytherapy, patients continue to suffer from adverse effects of this treatment modality. Significant morbidity can ensue from radiation therapy months to years after treatment. One specific adverse effect of radiation treatment for head and neck cancer patients is osteoradionecrosis (ORN) of the mandible. Being cured of their head and neck cancers, patients with advanced mandibular ORN may still succumb to extensive microvascular reconstructive surgery once reserved initially as an oncologic treatment option. These heroic measures may still prove futile because ORN can recur and little is still known about the disease.
ORN of the mandible is a pathologic condition that has been commonly described by clinical criteria, whereas a true definition has been elusive as the pathogenesis of this disease still remains undefined. Over the past 35 years, several authors have attempted to define ORN. Beumer et al stated that ORN occurs “when bone in the radiation field [is] exposed for at least 2 months in the absence of local neoplastic disease.” Marx defined ORN as “an area greater than one centimeter of exposed bone in a field of irradiation that had failed to show any evidence of healing for at least 6 months.” Hutchinson defined ORN to only require 2 months of exposed bone, whereas Harris used the 3-month marker.
Despite the variability in these clinical descriptions, several commonalities are present in clinically defining ORN: (1) exposed bone for at least 2 to 6 months, (2) a history of radiation therapy to the region of exposed bone, (3) the presence of necrotic or devitalized bone, and (4) no evidence of tumor recurrence. Patients with mandibular ORN often have radiologic assessment in the form of computed tomography (CT) and/or panorex. Limitations in radiologic analysis of ORN are not due to technology but again more so due to lack of defined radiologic criteria for mandibular ORN. Common radiographic findings for mandibular ORN include but are not limited to radiolucencies, bony sequestration, and/or pathologic fracture(s). Posttreatment histopathologic analysis often shows chronic inflammation and necrotic bone, yet the pathogenesis is unclear.
To investigate mandibular ORN further, we set out to first create an animal model as a platform to demonstrate the aforementioned criteria used today. Previous attempts at creating a model of ORN in a rat have been successful to some degree. However, these models were lacking in certain respects. Most of the models involved whole head irradiation, and this is not analogous to current radiation treatment for head and neck cancer. Recently, Niehoff et al described using HDR brachytherapy to create an experimental model of radiogenic bone damage in a rat. The HDR brachytherapy is a technique that uses a relatively intense source of radiation, typically iridium-192, to deliver a therapeutic dose of radiation through temporarily placed catheters at a specified location such as a unilateral rat mandible. The authors successfully demonstrated mandibular bone damage after HDR radiation. Mandibular ORN, however, was not described or defined.
It is known that dental trauma, especially tooth extractions in a radiated field, can increase the incidence of ORN . Several previous animal models involved maxillary tooth extraction, rather than mandibular. This is not as clinically relevant because it is well established that ORN predominately occurs in the mandible and not in the maxilla . Most studies used histologic examination of the specimens as the only evaluation tool. In reality, ORN is characterized by clinical examination with the aid of histologic and radiologic assessments.
The goal of our study was to improve and update the previous models of ORN not only using the most current technology, but incorporating the current knowledge of the disease process in our model. The rat model that has been designed involves irradiating rat mandibles with an extremely targeted device, extracting mandibular molars after radiation, and assessing outcomes using high-speed digital photography, micro-CT, and fluorescent and basic microscopy for histologic analysis.
A thoroughly effective treatment plan with minimal morbidity does not exist for ORN and most likely stems from a lack of understanding of the pathophysiology of the disease process. It is for this reason that we have attempted to create an updated rat model of ORN. Our hope is to establish a reproducible and clinically relevant animal model of post–dental extraction radiogenic bone damage from which to define and study the pathogenesis of mandibular ORN.
2
Materials and methods
2.1
Experimental design
Approval for the research protocol was obtained from the University of California Los Angeles Chancellor’s Animal Research Committee. Ten male Sprague-Dawley rats of 7 weeks of age (192–224 g) were used in this study. Rats were obtained from Charles River Laboratories International, Inc (Wilmington, MA). The rats were kept in pairs and given a standard pelleted rodent diet and water ad libitum in accordance with the requirements of the United States of America Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
The 10 rats were divided into 2 groups: group 1—left mandibular irradiation followed by left molar extractions (n = 6) and group 2—left mandibular sham irradiation (placement of HDR catheter only) followed by left molar extractions (n = 4).
2.2
Irradiation
Under inhalational isoflurane anesthesia, the left cheek skin of the rat was shaved and prepared with betadine. A sterile plastic HDR catheter (Alpha Omega Services, Long Beach, CA) was implanted along the lateral aspect of the left mandibular body. The catheter was inserted via a stab incision made just lateral to the left central incisor in the gingivolabial sulcus. The catheter was advanced submucosally along the inferior edge of the mandible. The catheter exited the oral cavity via a second stab incision at the posterior border of the mandibular ramus and advanced just inferior to the external auditory meatus. This advancement of the external portion of the catheter was consistently 10 mm and served as a reproducible length along with the above-mentioned landmarks. The external distal portion of the catheter was secured to the non–hair-bearing neck and cheek skin using Steri-Strip adhesive skin closure tape (3M, St Paul, MN) ( Fig. 1 ).
A 3-dimensional treatment plan for irradiation was devised that encompassed a depth of 5 mm from a point 1 cm from the distal aspect of the catheter tip. The center of the radiation field was placed lateral to the midline of the left mandibular body. A single dose of radiation was applied with an HDR afterloading remote machine (GammaMed 12it; Varian Medical Systems, Charlottesville, Inc, VA) providing 30 Gy with predefined isodose lines ( Fig. 2 ).
The catheter was removed after irradiation was complete in group 1 and immediately removed after placement in group 2 without radiation.
2.3
Tooth extraction
Under inhalational isoflurane anesthesia, all rats underwent atraumatic extraction of all 3 of the left mandibular molars 1 day following irradiation (or sham catheter placement). Extreme care was taken to avoid breaking the tooth roots from the crown. The teeth were examined after extraction with 3.5× surgical magnifying loupes to evaluate for completeness of extraction. Postoperative pain management was achieved with buprenorphine (Buprenex; Reckitt Benckiser Healthcare Ltd, Hull, England) at a dose of 0.03 mg/kg given subcutaneously. The diets were supplemented with sliced apples for the first 3 days after molar extractions.
2.4
Fluorochrome labeling
Sequential different-colored fluorochrome labeling of mineralizing bone was performed . Two fluorochromes were used in this experiment: calcein (green fluorescence) (1% in 2% NaHCO 3 solution, 20 mg/kg body weight; Sigma Aldrich, St Louis, MO) and demeclocycline (orange fluorescence) (1% in 2% NaHCO 3 solution, 20 mg/kg body weight; Sigma Aldrich). All rats underwent intraperitoneal injection of calcein 2 days before irradiation and demeclocycline 2 days before being killed. Fluorescent microscopy using UV light (Olympus BX51 Research Microscope, Tokyo, Japan) was used to analyze bone apposition at mandibular tooth sockets.
2.5
Killing of animals
The animals were killed 28 days post–tooth extraction. Mandibles were extracted and photographed using a Canon Rebel Ti DSLR camera (Canon, Tokyo, Japan). Mandibles were then placed in 70% isopropyl alcohol.
2.6
Radiologic analysis
CT of the extracted mandibles was performed using a desktop cone-beam micro-CT scanner ( μ CT 40 Scanco Medical, Brüttisellen, Switzerland). Three-dimensional reconstructions and volume analysis of the microradiographs were accomplished using μCT Evaluation Software v6.0 (Scanco Medical). The mandibular volume that was analyzed encompassed the region from the most superior aspect of the tooth socket where both medial and lateral cortices were seen (in an axial view) down to the nadir of the incisor root. Because of the relatively large volume that the incisor root socket normally encompasses in a rat mandible, these sockets were subtracted from volume calculations to limit the focus to the molar teeth regions. Bone volume (BV) and total volumes (TVs) were measured, and the ratio of these numbers was analyzed.
2.7
Histologic analysis
After micro-CT analysis was performed, both the radiated and control specimens were randomized into 2 groups to allow for non–decalcified-based fluorochrome analysis and decalcified-based paraffin embedded analysis, respectively. The first group consisted of 4 nondecalcified specimens (2 irradiated and 2 controls). These samples were taken from 70% isopropyl alcohol and immediately embedded in methyl methacrylate. Five-micrometer–thick sagittal sections of left hemimandibles were cut on a Jung polycot microtome (Reichert-Jung, Heidelberg, Germany) and either stained with toluidine blue or left unstained for fluorochrome analysis. Bone formation and resorption were analyzed at 2 standardized sites using a Pyser-SGI 5-mm stage micrometer scale (Pyser-SGI, Kent, United Kingdom) and an infinity eyepiece with dimensions of 10 mm by 0.1 mm divisions (Fisher Scientific, Pittsburgh, PA) at 20× magnification. Bone mineralization was detected via fluorescent microscopy in the center of the tooth extraction socket immediately above inferior alveolar nerve at a standardized location. Bone mineral apposition rate (MAR) was calculated along the lower cortical rim of the mandible directly below the posterior molar extraction socket and the inferior alveolar nerve at a second standardized location. All fluorochrome analysis was performed using an Olympus BX51 Research Microscope.
The 5 remaining specimens (3 irradiated and 2 controls) were decalcified in 10% EDTA (pH 7.4) for 10 days, fixed in formalin overnight, and paraffin embedded by the UCLA Translational Pathology Core Laboratory. Four-micrometer sagittal sections of the left hemimandibles were stained with hematoxylin and eosin (H&E) and viewed for qualitative histologic analysis using camera-assisted light microscopy (Nikon Labophot-2; Nikon, Tokyo, Japan).
2
Materials and methods
2.1
Experimental design
Approval for the research protocol was obtained from the University of California Los Angeles Chancellor’s Animal Research Committee. Ten male Sprague-Dawley rats of 7 weeks of age (192–224 g) were used in this study. Rats were obtained from Charles River Laboratories International, Inc (Wilmington, MA). The rats were kept in pairs and given a standard pelleted rodent diet and water ad libitum in accordance with the requirements of the United States of America Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
The 10 rats were divided into 2 groups: group 1—left mandibular irradiation followed by left molar extractions (n = 6) and group 2—left mandibular sham irradiation (placement of HDR catheter only) followed by left molar extractions (n = 4).
2.2
Irradiation
Under inhalational isoflurane anesthesia, the left cheek skin of the rat was shaved and prepared with betadine. A sterile plastic HDR catheter (Alpha Omega Services, Long Beach, CA) was implanted along the lateral aspect of the left mandibular body. The catheter was inserted via a stab incision made just lateral to the left central incisor in the gingivolabial sulcus. The catheter was advanced submucosally along the inferior edge of the mandible. The catheter exited the oral cavity via a second stab incision at the posterior border of the mandibular ramus and advanced just inferior to the external auditory meatus. This advancement of the external portion of the catheter was consistently 10 mm and served as a reproducible length along with the above-mentioned landmarks. The external distal portion of the catheter was secured to the non–hair-bearing neck and cheek skin using Steri-Strip adhesive skin closure tape (3M, St Paul, MN) ( Fig. 1 ).
A 3-dimensional treatment plan for irradiation was devised that encompassed a depth of 5 mm from a point 1 cm from the distal aspect of the catheter tip. The center of the radiation field was placed lateral to the midline of the left mandibular body. A single dose of radiation was applied with an HDR afterloading remote machine (GammaMed 12it; Varian Medical Systems, Charlottesville, Inc, VA) providing 30 Gy with predefined isodose lines ( Fig. 2 ).
