Evaluation of inferior alveolar nerve regeneration by bifocal distraction osteogenesis with retrograde transportation of horseradish peroxidase in dogs

Abstract

Background: Bifocal distraction osteogenesis has been shown to be a reliable method for reconstructing segmental mandibular defects. However, there are few reports regarding the occurrence of inferior alveolar nerve regeneration during the process of distraction. Previously, we reported inferior alveolar nerve regeneration after distraction, and evaluated the regenerated nerve using histological and electrophysiological methods. In the present study, we investigated axons regenerated by bifocal distraction osteogenesis using retrograde transportation of horseradish peroxidase in the mandibles of dogs to determine their type and function.

Methods and findings: Using a bifocal distraction osteogenesis method, we produced a 10-mm mandibular defect, including a nerve defect, in 11 dogs and distracted using a transport disk at a rate of 1 mm/day. The regenerated inferior alveolar nerve was evaluated by retrograde transportation of HRP in all dogs at 3 and 6 months after the first operation. At 3 and 6 months, HRP-labeled neurons were observed in the trigeminal ganglion. The number of HRP-labeled neurons in each section increased, while the cell body diameter of HRP-labeled neurons was reduced over time.

Conclusions: We found that the inferior alveolar nerve after bifocal distraction osteogenesis successfully recovered until peripheral tissue began to function. Although our research is still at the stage of animal experiments, it is considered that it will be possible to apply this method in the future to humans who have the mandibular defects.

Figure 1. Clinical image showing surgical procedures.

The IAN was not cut on the proximal side of the transport segment. Bifocal distraction was begun at a rate of 1/day and continued for about 10 days until the transport bone segment contacted the distal segment.

Figure 2. Extirpated mandibular bone.

After removing the reconstruction plate and distractor, the transport bone segment was in contact with the distal segment and generated callus was observed on the proximal side of the transport segment.

Figure 3. Histologic findings in total area at 3 months and 6 months (hematoxylin-eosin staining).

Histologic findings showed continuous nerve tissue from the proximal to distal side in all groups. Control: (flip horizontal image). The IANs were observed longitudinally from proximal side to distal side. 3 months: Fibrous connective tissue containing a number of blood capillaries was apparent in the nerve connection area. 6 months: Compared with that of 3 months, blood capillaries in fibrous connective tissue of the nerve connection area was decreased. Scale bar = 1000 µm.

Figure 4. HRP-labeled neurons (TMB staining).

HRP-labeled neurons were frequently seen from the base of the mandible nerve to the posterolateral area on both sides of the TG. A. TG on control side. B. HRP-labeled neurons on control side. C. TG after 3 months. D. HRP-labeled neurons after 3 months. E. TG after 6 months. F. HRP-labeled neurons after 6 months. TN: trigeminal nerve, Mx: maxillary nerve, Mn: mandibular nerve Scale bar = 1000 m in A, C, E, and 50 m in B, D, F.

Figure 5. Average number of HRP-labeled neurons per section.

The number of HRP-labeled neurons per section was low at 3 months and then increased to about 60% of the control side at 6 months.

Figure 6. Frequency of distribution of HRP-labeled cell body diameters.

The cell body diameter of HRP-labeled neurons on the control side ranged from 20∼70 µm (n = 400), with the majority sized from 37∼52 µm. After 3 months, they ranged from 26∼65 µm (n = 20), with the majority sized from 21∼36 µm, and after 6 months ranged from 19∼68 µm (n = 95), with the majority sized from 21∼36 µm. The size became reduced in the regenerated nerves.