Cyclophosphamide-Induced Morphological Changes in Dental Root Development of ICR Mice

Abstract

Background: Survivors of childhood cancer are at risk of late dental development. Cyclophosphamide is one of the most commonly used chemotherapeutic agents against cancer in children. The aim of this study was to investigate the effects of cyclophosphamide on root formation in the molars of growing mice and to assess the morphological changes in these roots using three-dimensional structural images.

Methods: We treated 16 12-day-old ICR mice with cyclophosphamide (100 mg/kg, i.p.) and 16 control mice with saline. At 16, 20, 24, and 27 days of age, the mandibular left first molars were scanned using soft micro-computed tomography. After scanning, the structural indices were calculated using a three-dimensional image analysis system, and the images were subjected to three-dimensional reconstruction. The length and apical foramen area of all distal roots were assessed. Histological changes in the apical region were then assessed via hematoxylin and eosin staining.

Results: The mandibular molars of all experimental mice showed evidence of cytotoxic injury, which appeared in the form of anomalous root shapes. Although all roots developed further after cyclophosphamide injection, the three-dimensional structural images showed that the roots in the experimental group tended to develop more slowly and were shorter than those in the control group. At 27 days of age, the mean root length was shorter in the experimental group than in the control group. Conversely, the apical foramen of the roots in the experimental group tended to close faster than that of roots in the control group. In addition, hematoxylin and eosin staining of the distal roots in the experimental group showed increased dentin thickness in the apical region.

Conclusion: Our results suggest that cyclophosphamide can result in short root lengths and early apical foramen closure, eventually leading to V-shaped or thin roots.

Fig 1. Points used for measuring root length for histological analysis.

Hematoxylin and eosin staining shows the distal root of the mandibular first molars at PN16; m, midpoint; c, cervical side; a, apical side. Scale bar = 100 μm. Original magnification, ×4.2.

Fig 2. Three-dimensional (3D) structure of the mandibular first molar roots.

Micro-computed tomography (micro-CT) images show buccal and apical views of the extracted mandibular left first molars through computerized 3D reconstruction.

Fig 3. Lengths of the distal roots of the mandibular left first molars.

The results are expressed as the mean ± standard deviation (SD). *Statistically significant difference compared with the control (t-test; p < 0.05).

Fig 4. Apical foramen areas in the distal roots of the mandibular first molars.

The results are expressed as the mean ± standard deviation (SDs). *Statistically significant difference compared with the control (t-test; p < 0.05).

Fig 5. Development of the apical region in the distal roots of the mandibular first molars.

Hematoxylin and eosin staining shows the distal root of the mandibular first molars in the control (A–D) and CY groups (E–H). HERS, Hertwig’s epithelial root sheath; DP, dental pulp; OD, odontoblasts; PDL, periodontal ligament. Scale bar = 100 μm. Original magnification, ×20.