Repairing Effects Of Olfactory Ensheathing Cells Combined With PLGA Conduit On Rat Sciatic Nerve Defect

With the development of modern microsurgery, the repair of peripheral nerve injury has made great progress in the recent yeas, but the results of the repair are still not satisfactory, especially for the peripheral nerve defect caused by severe trauma, resection of tumor and congenital deformity, which are difficult to be repaired and reconstructed in clinic at the present. The first candidate for clinically dealing with peripheral nerve defect is nerve autograft, but it is limited in the clinical practice on account of the shortage of available graft nerve, functional incapacitation of the donor area, limitation of bridging length, neuroma and spontaneous neuropathic pain resulted from the sacrifice of a healthy nerve. With the continuous development of synthetic material and bioengineering, the tissue-engineering artificial nerves give us a new hope to deal with the peripheral nerve defect satisfactorily. Olfactory ensheathing cells (OECs) belong to a distinct glial cell type and possess the capability of promoting axonal outgrowth and myelination. Poly(DL-lactide-co -glycolide) (PLGA) is a kind of biodegradable synthetic material, and is thought to be a promising artificial nerve scaffold material due to its advantage in rich source and good biocompatibility and plasticity. Based on above mentioned facts, by using PLGA conduit filled with the mixture of freshly isolated OECs and extracellular matrix gel, the 10mm sciatic nerve gap in rat model was bridged in this study, aiming at seeking a promising method to repair peripheral nerve defect and extending the nerve repair theory.In part one, by using a slightly modified method described in Sasaki et al 2004, OECs were isolated from the olfactory nerve layer and the glomerulor layer of olfactory bulb, and seeded on poly-L-lysine-coated plate. OECs were identified by immunocytochemical labeling for S-100 and NGFRp75 at 3 days after culture, and the purity of OECs was determined. In part two, the assessments of PLGA's compatibility with OECs were carried out at different set time by phase contrast inverted microscope, scanning electron microscope (SEM), MTT assay and flow cytometry. Then a 10 mm rat sciatic nerve defect model was eatablished and repaired with PLGA conduit filled with the mixture of CM-DiI pre-labeled OECs and extracellular matrix gel. At the same time, OECs+silicone, PLGA and silicone were used as the control. Cell tracing, functional evaluation, neurohistological assessment of regenerated nerve, target tissue measurement and neuron counting of spinal cord anterior horn were performed after surgery for detecting the repair effects of OECs combined with PLGA conduits on sciatic nerves and downstream target tissues as well as protective effects on upstream neurons. The results are as following:1. A stable method for OECs isolation and culture was established. Under phase contrast inverted microscope, OECs were bipolar or multipolar cells with a clear outline, and characteristically presentd by their thin and long processes, and positively immunostained for NGFRp75 and S-100 protein. The purity of isolated OECs was between 50% and 60% .2. Observation of phase contrast inverted microscope and SEM showed no difference about OECs'morphological feature between PLGA group and control group. MTT results revealed PLGA had no deleterious effect on metabolic activity of OECs. There was no statistically difference of the cell cycle between experimental group and control group. Furthermore, the DNA index values of OECs in PLGA group were normal, indicating normal diploid cells. PLGA did not impose carcinogenicity on OECs.3. CM-DiI labeled OECs could survive up to 6 weeks after surgery and distributed longitudinally along the nerve. In addition, OECs'survival time in the OECs+PLGA group was longer than that in OECs+silicone group.4. At 8 weeks after surgery, the OECs+PLGA group had a recovery rate of heel ulcer of 83.3%, a relative diameter recovery rate of 58.0±2.9%, and without autophagy phenomenon, indicating better results in general state than other three groups. The bridging PLGA conduit possessed nerve-like appearance, with some superficial blood supply on its surface and no adhesion with surrounding tissues. Contrary to PLGA conduit, silicone conduit had a obvious connective membrane on its surface without blood supply.5. First sign of sensory recovery could be monitored at 6 weeks after surgery in OECs+ PLGA group with the retraction time of 3.05±0.11sec, which was significantly shortened compared to three other groups ( P<0.05). At 8 weeks after surgery, the sensory recovery in OECs + PLGA group continued,and at the same time, the retraction time in OECs+silicone group was decreased to 3.06±0.09 sec, showing a better improvement of sensory function in OECs+PLGA group than that in other groups without OECs treatment ( P<0.05). Through 8 weeks after surgery, there was no any improvement of motor function indicated by sciatic function index (SFI) in every group. The OECs+PLGA group with a shortest latency period of 3.10±0.11ms and a fast nerve conduction velocity of 19.38±0.70 m/s demonstrated significant better results than other three groups(P<0.05). Furthermore, there was a very significantly higher amplitude of compound muscle active potential in OECs+PLGA group compared to three other group(P<0.01).6. At 8 weeks after surgery, the recovery rate of gastrocnemius muscle water weight in OECs+ PLGA group was 67.22±4.46%, and significantly higher than that in other three groups. In addition, OECs+PLGA group had a motor endplate number of 55.5±3.7, a major axis length of 41.5±8.8μm and a minor axis length of 22.5±3.8μm, indicating better results than other three groups( P<0.05).7. At 8 weeks after surgery, there were positive neurons observed by DiI retrograde tracing in all four groups. Percentage of neuron number in experimental side / neuron number in normal side was 77.1±3.0%in OECs+PLGA group. This data indicated that there was no significant difference between OECs+PLGA group and OECs+silicone group(P>0.05), but a significant difference between OECs+PLGA group and other three groups(P<0.05). In addition, the number of large-sized and medium-sized motoneurons in lateral of spinal cord anterior horn was counted. The number of motoneurons in OECs+ PLGA group and OECs+ silicone group was 9.2±0.8 and 9.2±0.4 respectively, and significantly higher than that in other two groups without OECs treatment(P<0.05).8. At 8 weeks after surgery, the toluidine staining showed the fiber density of regenerated nerve middle segment in OECs+PLGA group was 17713±2505/mm2, and significantly higher than that in other three groups(P<0.05). The total nerve fiber number in OECs+ PLGA group was 6627±1032, and significantly better than other two groups without OECs treatment (P<0.05), but there was no difference between OECs+PLGA group and OECs+ silicone group (P>0.05). In the distal segment of regenerated nerve, the fiber density and the total nerve fiber number of OECs+PLGA group and OECs+silicone group were respectively higher than those of PLGA group and silicone group. At the same time, transmission electron microscope (TEM) observation revealed that the maturity of myelinated nerve fiber in OECs+ PLGA group was highest, and the number of unmyelinated nerve fiber in this group was least compared to other three groups. The above results suggest that:1. PLGA has no deleterious effects on morphological feature, proliferative capacity and cell cycle of OECs, so it processes good compatibility with OECs, and is suitable to construct tissue-engineering artificial nerve with OECs.2. CM-DiI labeled OECs can survive in the bridging conduits after transplantation, and PLGA conduit can support the survival of transplanted OECs.3. The combination of OECs and PLGA can improve the recovery of gastrocnemius muscle water weight, and promote the reconstruction of the motor endplates, indicating this combination can improve the reinnervation of target tissues.4. The combination of OECs and PLGA can promote reconnection between regenerated nerve fibers and related neurons , and the survival of the large-sized and medium-sized motoneurons in lateral of spinal cord anterior horn as well.5. The combination of OECs and PLGA can promote the recovery of sensory function and electrophysiological function , the axonal outgrowth and myelination.