Rat Facial Nerve Regeneration with Human Immature Dental Pulp Stem Cells

Abstract

Facial paralysis can result in severe implications for the patients. However, stem cell biology has become an important field in regenerative medicine since the discovery and characterization of mesenchymal stem cells. Our aim was to evaluate the regeneration after facial nerve crush injury and application of human immature dental pulp stem cells (iDPSC). For this study 70 Wistar rats underwent a unilateral facial nerve crush injury and were divided into two groups: Group I (GI): Crushed; Group II (GII): Crushed and iDPSC, and distributed into study periods of 3, 7, 14, 21, and 42 postoperative days. Facial nerve regeneration was analyzed via functional recovery of whisker movement, histomorphometric analysis, and immunoblotting assay. The results show that GII had complete functional recovery at 14 days, while GI recovered after 42 days. Also, regarding the facial nerve trunk, GII presented histological improvement, evidencing better axonal and structural organization of the myelin sheath, and exhibited statistically higher values for the outer and inner perimeters and g-ratio. Nevertheless, GI exhibited statistically higher values for the thickness of myelin sheath. In the buccal branch, no differences were observed for all parameters between groups. At 42 days, both groups GI and GII were close to the levels observed for the control group. Concerning nerve growth factor expression, GII exhibited statistically greater values (p < 0.05) compared with the control group at 7 days. In summary, a single injection of human iDPSC promoted a positive effect on regeneration of the facial nerve trunk after 14 days and provided an alternative to support regeneration following peripheral nerve injury.

Keywords: deciduous teeth; facial nerve; nerve injuries; stem cells.

Figure 1.

Cross-section TEM images of the facial nerve trunk for the GC, GI, and GII groups on 3, 7, 14, 21, and 42 days (2,500× magnification). (*) Myelin disruption; (Md) Myelin debris; (arrow) Myelin sheath infolding; (star) recent demyelination; (NM) Non-myelinated nerve fibers; (UA) Unmyelinated axon; (C) Connective tissue; (RA) Remyelinated axon. Scale Bar = 10 µm.

Figure 2.

Cross-section TEM images of the facial nerve trunk for the GC, GI, and GII groups on 3, 7, 14, 21, and 42 days for ultra-structural analysis (20,000× magnification). (Md) Myelin debris; (arrow) Myelin sheath infolding; (Ax) Myelinated axon; (SC) Schwann cell. Scale Bar = 02 µm.

Figure 3.

Cross-section TEM images of the facial nerve trunk for the GC, GI, and GII groups on 3, 7, 14, 21, and 42 days for ultra-structural analysis (100,000× magnification) of lamellae of the myelin sheath. Scale Bar = 0.2 µm.

Figure 4.

Cross-section TEM images of the buccal branch for the GC, GI, and GII groups on 3, 7, 14, 21, and 42 days (2,500× magnification). (Md) Myelin debris; (arrow) Myelin sheath infolding; (Ax) Myelinated axon; (UA) Unmyelinated axon; (Ad) axon degeneration; (RA) Remyelinated axon. Scale Bar = 10 µm.

Figure 5.

Cross-section TEM images of the buccal branch for the GC, GI, and GII groups on 3, 7, 14, 21, and 42 days for ultra-structural analysis (20,000× magnification). (arrow) Myelin sheath infolding; (Ax) Myelinated axon; (My) Myelin sheat; (C) Connective tissue; (SC) Schwann cell. Scale Bar = 02 µm.

Figure 6.

Cross-section TEM images of the buccal branch for the GC, GI, and GII groups on 3, 7, 14, 21, and 42 days for ultra-structural analysis (100,000× magnification) of lamellae of the myelin sheath. Scale Bar = 0.2 µm.

Figure 7.

Statistical analysis of the g-ratios of the facial nerve trunk. The g-ratio was significantly improved in the GII group relative to the GI group (^# p < 0.05).

Figure 8.

Statistical analysis of the number of myelinated fibers of the facial nerve trunk. There was no statistical difference between groups.

Figure 9.

Statistical analysis of the g-ratios of the buccal branch. There was no statistical difference between groups.

Figure 10.

Statistical analysis of the number of myelinated fibers of the buccal branch. There was no statistical difference between groups.

Figure 11.

The expression levels of NGF to the facial nerve trunk for the GC, GI, and GII groups at all postoperative time. There were differences between the groups GII and GC at 7 postoperative days (*p < 0.05).