Facial nerve defect can severely affect patients’ quality of life.1 Treatments include surgical reinnervation procedures (end-to-end anastomosis), autologous nerve grafts, etc. However, many treatments have severe side effects, and the facial motor function can hardly return to normal after surgery.2

Peripheral nerves do have the potential to regenerate after injury. Current strategies to repair injured peripheral nerve system focus on developing bridging scaffolds that could guide axonal regeneration across the lesion site. With the development of tissue engineering, it is possible to use nerve conduits, grafts filling material, and appropriate seed cells to bridge the injured peripheral nerve and to promote nerve regeneration.2 Previous studies showed that the outcome of peripheral nerve regeneration using tissue engineering is similar to that of autogenous nerve transplantation.3

Olfactory ensheathing cell (OEC) is a unique kind of glia cells, which can promote axon growth.4 OECs can maintain somatotopic organization after lesion, promote regeneration of olfactory nerve fibers, and improve the organization as well as the quantity and speed of nerve regeneration.5–7 Many studies showed that OECs transplantation has a promising therapeutic value in central and peripheral nerve injury, especially in spinal cord injury.6,8

Finding a reliable source of OECs which is easily accessible and can ensure a sufficient number of cells is a major prerequisite for conducting studies on OEC-mediated nerve regeneration. Most previous studies obtained OECs from the Olfactory Bulb (OB) within the cranial cavity.5 OECs can also be isolated from the Olfactory Mucosa (OM),9,10 which may provide a more readily available source of these cells for autologous transplantation. Previous studies showed that OECs obtained from the OM were therapeutic when transplanted into the injured Central Nerve System (CNS) or Peripheral Nerve System (PNS) of patients and animal models.11–15 In addition, there is no associated comorbidity since it has been shown that olfactory biopsies do not impair the sense of smell.16

Previous studies showed that OM-OECs and OB-OECs both have the capability to promote nerve regeneration, but little is known about their differences. In this study, we transplanted different originated OECs (OM-OECs and OB-OECs) in PLA/chitosan conduit to bridge the defected facial nerve in rat, and compared the differences between the function of inducing nerve regeneration of OM-OECs and OB-OECs. Behavioral, electrophysiology, histology, and retrograde tests were used to assess the outcomes.

OECs from rat OM and OB were successfully isolated and cultured, and highly purified and stable OECs were obtained. All rats survived after the operation.

Facial muscle function score

Immediately after a facial nerve lesion, all rats presented complete facial paralysis on the surgery side, with whisker-pad, failure to move the eyelids, sunken beards, and weakened wink reflection. Over the following weeks, facial function improved in all groups, but muscle movement remained worse compared with the healthy side (Fig. 2).

Figure 2.

Gross observation of the three groups of rats 4 and 8 weeks after the procedure.

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The facial function score in Group A, B and C, after 4 and 8 weeks, were shown in Table 1. After 4 and 8 weeks, there were significant differences in facial nerve function scores between Group A, B and C, and there were no differences between Group A and B (Table 1).

Table 1.

Facial function score (x±s).

Group (n=6)	4 weeks	8 weeks
A: OM-OECs	2.17±0.41	3.00±0.63
B: OB-OECs	2.00±0.63	2.83±0.75
C: DF12/FBS	1.17±0.41	1.83±0.41
Control	4	4
p-value (A vs. B)	0.30	0.34
p-value (A vs. C)	0.00088	0.0023
p-value (B vs. C)	0.01	0.01

Histological observationGross pathology

Four weeks after the surgery, nerve defects were repaired by regenerative nerve in all groups. The nerves were easy to separate without adhesion and scar formation. There was no neuroma formation in the proximal and distal end of regenerative nerve. PLA/chitosan conduits became thinner and easy to fracture, but still intact. Eight weeks after the surgery, PLA/chitosan conduits were partially absorbed; and the conduit walls were not intact. In Group A and B, the regenerative facial nerve had good continuity, without thinning and hardening. The appearances of the regenerative nerves were similar to normal (Fig. 3).

Figure 3.

The regenerative facial nerve in the conduit (a, conduit; b, the distal end of the facial nerve; c, regenerative nerve).

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Light microscopic findings

Normal facial nerve possesses the myelinated axons with thick uniform myelin layer, composed of one to five fascicles enveloped by epineuria with spindle shaped cells, and axons were regularly arranged and had similar diameters (original magnification 2000).

Four weeks after surgery, in Group A, myelinated axons with large diameter were nearly normal, but small-diameter myelinated axons were irregular. In Group B, occasional myelinated axon was presented with many Schwann cells with variable morphology. In Group C, there were a large amount of degenerated dark myelin clumps without axons (bungner bands) and severe fibrosis, and the perineurium was a condensation of loose connective tissue.

Eight weeks after surgery, the maturity of the regeneration nerve was better in all groups compared with that of 4 weeks. In Group A, regenerative nerve bundles were similar to normal nerves, with uniform size. Myelins were dense, with no deterioration. Schwann cells were well distributed, with similarly sized and shaped cell nuclei. In Group B, rich vessels were seen in the epineurium, bundled regenerative nerve fibers, and many neovessels in the nerve fiber bundles. In Group C, there were fewer regenerative axons with uneven distribution and poor development, and a lot of fibrous connective tissue (Fig. 4).

Figure 4.

The histological observation of the regenerative nerve in three groups under light microscopy.

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Electron microscopy findings

Table 3 shows the number of myelinated nerve fibers, the nerve fiber diameter, and the thickness of the myelin sheath in Group A, B and C, after 4 weeks and 8 weeks respectively.

Table 3.

Electron microscopy finds (x±s).

Group (n=4)	Number of myelinated nerve fibers		Diameter of nerve fibers (μm)		Thickness of myelin sheath (μm)
	4 weeks	8 weeks	4 weeks	8 weeks	4 weeks	8 weeks
A: OM-OECs	1911.50±78.10	2743.75±82.47	3.33±0.15	4.73±0.12	0.71±0.06	0.89±0.06
B: OB-OECs	1899.75±60.46	2319.50±91.83	3.21±0.16	3.96±0.12	0.69±0.05	0.75±0.02
C: DF12/FBS	1763.50±83.33	2008.50±134.10	2.39±0.06	3.10±0.03	0.40±0.04	0.55±0.02
Control	3022.50±133.75	3096.75±110.91	5.65±0.36	5.99±0.36	0.93±0.04	0.94±0.06
p-value (A vs. B)	0.41	0.00025	0.16	0.00005	0.31	0.0070
p-value (A vs. C)	0.021	0.00012	0.00017	0.000025	0.00014	0.00034
p-value (B vs. C)	0.021	0.0055	0.00041	0.00021	0.000066	0.0000039

After 4 weeks, the number of regenerative nerve fibers, the diameter of the fiber, and the thickness of myelin sheath in Group A and B are superior to that of Group C, and there were no differences between Group A and B. After 8 weeks, the number of regenerative nerve fibers, the diameter of the fiber, and the thickness of myelin sheath in Group A are superior to that of Group B, and that of Group B are superior to that of Group C (Fig. 5).

Figure 5.

The histological observation of the normal facial nerve the regenerative nerve in three groups under electron microscopy.

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Fluorescent retrograde tracing

Table 4 shows the average numbers of FG-labeled cells in the facial nucleus at 4 and 8 weeks after cell transplantation in Group A, B and C, respectively.

Table 4.

number of FG-labeled cells (x±s).

Group (n=2)	4 weeks	8 weeks
A: OM-OECs	175.00±18.38	237.50±12.02
B: OB-OECs	154.00±5.66	191.00±8.49
C: DF12/FBS	53.50±6.36	96.00±9.90
Control	271.50±20.51	279.50±12.02
p-value (A vs. B)	0.17	0.028
p-value (A vs. C)	0.0023	0.0034
p-value (B vs. C)	0.0019	0.0050

After 4 weeks, there were significant differences in FG-labeled cell number between Group A, B and C, and there was no differences between Group A and B. After 8 weeks, there were significant differences in FG-labeled cell number between Group A, B and C; A>B>C (p<0.01) (Table 4) (Fig. 6).

Figure 6.

The FG-labeled cells in the facial nucleus of the three groups of rats 4 and 8 weeks after the procedure.

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Glial cells transplantation has been frequently used to promote axonal regeneration in Central and Peripheral Nervous System (CNS PNS). Myelin-forming cells usually used for transplantation are Schwann cells, olfactory ensheathing cells, etc.18 OECs are unique glial cells that share some properties with astrocytes and Schwann cells but have some intrinsic properties. Studies showed that OECs could mediate neurotrophic and neuroprotective effects by secretion of chemokines, enhance axonal regeneration and produce myelin after transplantation.4 It has been observed that OECs can promote axonal regeneration of CNS and PNS neurons in culture and in vivo,6 which make them ideal candidates for cellular transplantation.

Olfactory Bulb (OB) is the most commonly used source to obtain OECs in most studies. However, it is not a clinically favored site for sourcing autologous OECs because of the morbidity associated with surgical access and destruction of the olfactory bulbs to obtain the cells. OECs can also be isolated from the olfactory mucosa (OM). Previous studies showed that OM-OECs also have the capability to promote nerve regeneration,13 but there are very few studies about the differences between OM-OECs and OB-OECs on their effect of nerve regeneration.

It is known that peripheral nervous system has the ability to achieve functional recovery after nerve injury, and rat facial neurons are able to bridge gaps of up to 10mm. There are some studies about different methods to facilitate facial nerve regeneration,19–24 and some studied OECs transplantation in facial nerve injury,23,24 but most of the studies focused on the collateral axonal branching of the regenerative facial nerve. Recently there is a study about human olfactory stem cells from OM could improve the recovery of rat facial nerve injury in two measurements: whisker movement and electroneuronography analyses.24 In our study, we try to use different measurements to better evaluate the nerve regeneration outcome of different oriented OECs transplantation after PNS injury.

Facial muscle function is the most objective result of nerve regeneration. The recovery of facial muscle function is the last step of nerve regeneration, which means the regenerative nerve has the capability to control the target muscle. After 4 and 8 weeks, there were significant differences in facial nerve function scores between Group A, B and C, and there was no differences between Group A and B. The result means that facial muscle functions were similar in Group A and B, and among the three groups, the rats in Group C had the worst facial function. It could be concluded from the results that both of OM-OECs and OB-OECs can stimulate the regeneration of injured facial nerve.

Facial nerve evoked potential is the most effective index to show the regeneration degree of the injured nerve. The improvement of the evoked potentials means that the regenerative nerve was able to conduct action potentials to the target tissue. After 4 weeks, there were significant differences in latency and amplitude between Group A, B and C, and there was no difference between Group A and B. After 8 weeks, latency A<B<C, amplitude A>B>C. Studies showed that facial nerve evoked potential is the first sign to show change after effective nerve regeneration, while the muscle function is the last to show the change,25 which explains that in our study, after 8 weeks, there were significant differences in the latency and amplitude among Group A, B and C, but there was no differences between Group A and B in facial muscle function scores.

Histological observation gives us a direct observation of the nerve regeneration. Light microscopic findings shows that the regeneration in Group A was better than Group B, and Group B was better than Group C. After 8 weeks, the regenerative nerves in Group A were similar as the normal ones. We calculated the values of the number of the nerve fibers, the fiber diameter of the myelin sheath, and the sickness of myelin sheath in three groups under electron microscopy. The results were in accordance with the findings under light microscopy. These histological observations revealed that, the OECs transplantation can lead to nerve tissue regeneration with large numbers of myelinated nerve fibers, crude fibers, and larger myelin thickness and volume in the transplanted graft, and OM-OECs transplantation seemed to have better results in long term.

Fluorescent retrograde tracing results showed that whether the regenerative nerves had set up the efficient connection to the facial nucleus. The histological structure of the regenerative nerve connecting two ends of injured nerve is only the first step for complete functional recovery. It does not mean the regenerative nerve has achieved complete functional restoration. Only the regenerative nerve has efficient connection with the facial nucleus, means the whole nerve pass is set up.26 In this study we counted the FG-labeled cells in the facial nucleus at 4 and 8 weeks after cell transplantation. The results revealed that OECs transplantation stimulated the connection between regenerative nerve and facial nucleus, and the transplantation of OM-OECs had a better effect of nerve connection compared with OB-OECs in the long term.

In this study, the facial nerve regeneration was evaluated in four different ways: functional assessment of the terminal organ, electrophysiology, histology (qualitative and quantitative), assessment of whole nerve pass way connection. Almost all the four evaluations had similar results: OECs could promote the regeneration of injured facial nerve. Compared with OB-OECs, OM-OECs were more effective in promoting nerve regeneration, and this difference was more significant 8 weeks after the transplantation than 4 weeks.

These results showed that OECs transplantation with nerve conduits may acts as a powerful tool to enforce peripheral nerve regeneration under adverse conditions, e.g., long deficit between the two ends, and OM-OECs could be a more convenient and effective source for transplantation. There are still remaining questions that need to be addressed further. For instance, postparalytic syndrome (abnormally associated movements after facial nerve transaction) is caused by a given muscle group reinnervated by misrouted axonal branches. Olfactory ensheathing cells have been shown to reduce axonal sprouting and stimulate axonal regeneration after transplantation into the spinal cord. Could OECs transplantation also reduce sprouting of a damaged peripheral pure motor nerve? Is OECs transplantation more effective compared with simple facial-facial anastomosis? What is the mechanism of the transplanted OECs promoting nerve regeneration?

We discovered that OECs with nerve conduit could improve the recovery of injured facial nerve, and OM-OECs are more effective for facial nerve regeneration compared with OB-OECs 8 weeks after the transplantation. These results could cast new light on finding new treatment for facial nerve defect, and furnish the foundation of auto-transplantation of olfactory mucosa OECs in periphery nerve injury. Larger studies are needed to confirm our findings and unravel the underlying mechanisms.

This work was supported by National Natural Science Foundation of China (no. 81300837).

The authors declare no conflicts of interest.