Development of a Preclinical Double Model of Mandibular Irradiated Bone and Osteoradionecrosis in New Zealand Rabbits

Abstract

Purpose: Radiotherapy (RT) plays a crucial role in head and neck (HN) cancer treatment. Nevertheless, it can lead to serious and challenging adverse events such as osteoradionecrosis (ORN). A preclinical rabbit model of irradiated bone and ORN is herein proposed, with the aim to develop a viable model to be exploited for investigating new therapeutic approaches.

Methods: Nine New Zealand white rabbits were irradiated using a single beam positioned to the left of the mandible and directed perpendicular to the left mandible. A 10 × 10 mm² region of interest (ROI) located below the first molar tooth on the left side was identified and irradiated with 7 Gy each fraction, once every 2 days, for five fractions. Dose distributions demonstrated that the corresponding ROI on the contralateral (right) mandibular side received approximately 5 Gy each fraction, thus bilateral irradiation of the mandible was achieved. ROIs were categorized as ROI_H on the left side receiving the high dose and ROI_L on the right side receiving the low dose. Rabbits were followed up clinically and imaged monthly. After 4 months, the irradiated bone was excised, and histological examination of ROIs was performed.

Results: Radiological signs suggestive for ORN were detected in the entire population (100%) 16 weeks after irradiation on ROI_H, which consisted of cortical erosion and loss of trabeculae. ROI_L did not show any radiological evidence of bone damage. Histologically, both sides showed comparable signs of injury, with marked reduction in osteocyte count and increase in empty lacunae count.

Conclusions: A preclinical double model was successfully developed. The side receiving the higher dose showed radiological and histological signs of bone damage, resulting in an ORN model. Whereas the contralateral side, receiving the lower dose, presented with histological damage only and a normal radiological appearance. This work describes the creation of a double model, an ORN and irradiated bone model, for further study using this animal species.

Keywords: experimental animal models; head and neck; mandibular irradiation; osteoradionecrosis; radiotherapy.

FIGURE 1

Study workflow. CE, contrast‐enhanced; CT, computed tomography; MR, magnetic resonance; H&E, hematoxylin–eosin staining. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Irradiated target area. Bidimensional target localized at the inferior border of the mandible, below the root of the first molar (the irradiated area is marked in black).

FIGURE 3

Dose distributions. The figure shows the irradiation dose distribution. The irradiation, sourced from the left of the animal, reached both the left and right mandibular bodies with decreasing dose. The dose distribution map revealed that the right mandibular body received approximately 5 Gy/fraction. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Radiologic signs of ORN on ROI_H over the study period. Three explanatory examples of development of radiologic signs of bone injury at the different timepoints: Pre‐RT (A), 4 weeks (B), 12 weeks (C), and 16 weeks (D) after irradiation. Coronal sections are displayed (in posteroanterior vision, left mandibular body correspond to the left of the image). Case 1: First signs of ORN consisted of loss of trabeculae (asterisk) and bone sclerosis (arrowhead) of the left mandible, detected 16 weeks after irradiation (1D). Case 2: First signs of ORN occurred 12 weeks after irradiation, with extended unicortical erosion on the lingual side of the left mandible (arrow) (2C); bone sequestrum was evident 16 weeks after irradiation (arrowhead) (2D). Case 3: Radiologic signs of bone damage were evident 4 weeks after irradiation (3B), with unicortical erosion on the lingual side (arrow) of the left mandible; bicortical erosion appeared 12 weeks after irradiation (arrows) (3C) and loss of trabeculae (asterisk) 16 weeks after the irradiation (3D).

FIGURE 5

Osteocytes and empty lacunae count. Description through histograms of the mean and 95% confidence interval (95% CI) of osteocytes (left) and empty lacunae (right) count in irradiated bone. No statistical differences were observed in terms of osteocytes and empty lacunae count between the high‐dose (35 Gy) and low‐dose (25 Gy) groups. ROI_H = region of interest irradiated with high dose; ROI_L = region of interest irradiated with low dose. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

Histological signs of postirradiation bone damage. (A) Histological section that shows bone marrow damage with initial loss of hematopoietic cells (arrows). (B) Example of hemorrhagic bone marrow surrounded by empty lacunae. (C) Bone marrow with initial signs of vessel hyalinization and loss of cellularity (arrows). (D) Diffuse damage of the bone matrix: Inhomogeneous necrosis of the matrix (circles) close to fibrosclerotic trabeculae (arrows). (E) Bone section with empty lacunae. [Color figure can be viewed at wileyonlinelibrary.com]