| Background and Objective:Peripheral nerve injury is very common in the field of modern surgery. It is accompanied with these factor such as trauma, disease, surgery and so on. The incidence rate is very high, and the result of the injury is related with the injury level of the nerve, could be physical disability or performance state. Attention with the result of injury, such as neonatal brachial injury and the root avulsion of brachial plexus injury, we can't repair it in such a higher range by end-to-end suture after resecting the damaged region. For repairing the higher range defect of the peripheral nerve, the classical method autograft as the "gold standard", on the one hand will produce additional surgical procedures, resulting in dysfunction for donor site; on the other hand derived from somatic limited, and it is diffcult to meet the need of long segement of nerve defect. Therefore, under the guidance of the principle of tissue engineering, micro surgeons and researchers through the unremitting efforts, the expectations of artificial nerve conduit to replace the autologous nerve graft.This study main to discover the possibility that repairing the rats'sciatic nerves defects of using the compound controlled releasing drugs PLGA-membranes of NGF and FK506 together with AECM. Materials and Methods:BSA(0.01g) as the carrier together with NGF(3000AU) and FK506(10mg) were compounded into distilled water, and then were dried in the Vacuum freeze-drying equipment. PLGA was dissolved in chloroform and configured to 10% solution, which was stired with magnetic stirrer when joined the aforementioned freeze-dried powder as the proportion PLGA 0.1g:FK506 10mg:NGF 3000AU:BSA 0.01g. The mixture solution was poured into the mold, using solvent evaporation method, to produce the membrances as the regulation of 12mm X 8mm X 0.3mm. The membrances were placed into ethanol solution and extracted the remaining chloroformd and monomer and oligomer in 2 hours. Then, flushing with Deionized water 5 min, the membranes were standby vacuumed to constant weight.The preparation of NGF, FK506/PLGA controlled releasing membrane placed into 1 ml PBS containing 1% BSA in the closed Ep tube, at 37℃for 48 hours. Then the membrane was releasing 1 hour in the new medium as before. Removed the composite membrance, and tested the quantity of NGF and FK506 released in the Ep tube by ELISA. After testing the membrane was placed and incubated into a new medium as before again, and for the ELISA test every three days.Reference the preparation method of Dument and Sondell Chemical extraction acellular nerve extracellular matrix and on this basis as part of the improvements. Specific steps are as follows:(1) the two sides sciatic nerves was cut about 1.2 cm, and stripped the connective tissue covered, then washed by Hank's solution 3 times; (2) put the nerve into 3% Triton-X-100 for soaking 24 hours, the washed by Hank's solution 3 times; (3) shocking the 4% sodium deoxycholate soaked the nerves in, and the nerves was cleaned by Hank's solution for washing 3 times; (4) repeat the steps as (2),(3); (5) the produced AECMs are stored in Hank's solution 4℃; (6) storage solution replaced weekly. The preparative AECMs are milky white and translucent.30 adult male SD rats, weight 200±20g, prepared for the 10 mm unilateral (left) sciatic nerve defect model, and randomly divided into 3 groups(n=10):Group A: repair the defects by NGF/FK506 compound PLGA-membranes together with the prepatated AECM; Group B:repair the defects by AECM only; Group C:repair the defects by the autologous nerves. Sodium pentobarbital (40mg/kg) intraperitoneal injection of anesthesia, have a left hind leg posterolateral incision, and expose the sciatic nerve. Resection sharp of the distal sciatic nerve 8mm lower the piriformis 3mm in Group A, B, and wait until the natural back reduction, produce the 10 mm rat sciatic nerve defect model.10-0 suture no damage under the microscope bridge closure with AECM first, and then in Group A, controlled-release membrane completely wrapped bridge area and interrupted suture. Group C lower next the piriformis 3mm sharp cut 10mm of the distal sciatic nerve, then according to blood vessels under the microscope in situ suture end to end.Compare the general observation results respectively at 4,8,12 weeks after operations, and at 12 weeks,compared the results by the number of nerve regeneration, nerve fibers arrangement, myelination, nerve electrophy-siology and histology.Have the statistics test with one-side variance analysis by SPSS 12.0, choosing statistical significance of P<0.05.Result:①In vitro, NGF can't be detected by ELISA after 18 days and FK506 about 28 days, the compound membranes were fully degraded after about 14 weeks. In vivo, the controlled releasing membranes start to degrade after 4 weeks, and have significant degrading after 8 weeks, and almost degraded at 12 weeks. It complies that the degrading time in vivo is less than in vitro, and the period of the compound membrane's degradation is basicly fit the period of the nerve regeneration in vitro and in vivo.②At 4 weeks and 8 weeks after operation, the nerve regeneration through the gap in Group A and C were better than the Group B, but the bridging zone adhesions in Group A and B were better than the Group C; Some toes movement could be observed in Group A and C, while the Group B was not found; At 12 weeks, the operation ipsilateral toes'ulcer healing and the function of the ipsilateral legs restored almost, and the situation of nerve regeneration number, arrangement, myelination and electrophysiological, histological about Group A and C were better than the Group B. ConclusionThe compound controlled releasing drugs of NGF and FK506 PLGA-membranes together with AECM can be the ideal bridging materials to repair the surround nerve defects, and provid the experimental basis for the cilinal peripheral nerve defects repairing study. |
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