The Construction And Application Research On Peripheral Nerve Defect Reparation Of The Compound Nerve Conduit

OBJECTIVE: To prepare NGF microspheres with high bioactive drug and continuous drug release behavior in three months by choosing suitable protectives and encapsulation material; To construct a compound nerve regeneration conduit and study its application in the repairment of peripheral nerve defect.METHODS: The microspheres with PEG, OVA, BSA and glucose as protectives were prepared by W/O/W emulsion based solvent evaporation method respectively. The microspheres that were encapsulated by PLGA of different viscosity and PLA were prepared by S/O/W emulsion based solvent evaporation method. NGF content was assayed by an enzyme-linked immunosorbent assay (ELISA), while its bioactivity was measured by PC12 cell method. The nerve conduits, which contained of different amount of additives were prepared with solvent evaporation method. Three kinds of compound nerve regeneration conduit were constructed to repair the rat sciatic nerve defect of 10mm: compound conduit A (PLA conduit filled with 10 mg microspheres), compound conduit B (PLA conduit with 10 mg microspherers surrounded) and compound conduit C (PLA conduit filled with 5 mg microspheres). The appropriate compound conduit was choosen by nerve regeneration results. After that, the effect of compound conduit in nerve regeneration was evaluated by rat sciatic nerve defect repairment. The rats were divided into four groups randomly, each was given one of the following repairment: A. the autogenetic nerve transplantation, B. PLA conduit with NGF microspheres, C. PLA conduit with physiological saline, D. the compound conduit with NGF solution. The regenerated length and quantity of nerve fiber were studied after 15 days. Nerve structure was observed by HE staining.RESULTS: The average recovery of NGF extracted from microspheres was 81.3%±6.2% with RSD 4.32% when assayed by ELISA. In the range from 3.9-250 pg/ml, the determination curve was y=0.0054x+0.1067, with r being 0.9908. When PC12 cell was used to measure the bioactivity of NGF, the standard determination equation was y=0.005x³-0.2612x²+5.1218x+14.20 with r being 0.9957, and the quantitive range was 1～30 ng/ml NGF. OVA was chosen as protective agent and PLA:PLGA(1:1) was used to prepare the objective microspheres. The average particle size was about 25μm, the encapsulation efficiency was 29.72% and the drug loading was 0.003%. The microspheres could release bioactive drug for three months continuously with a burst release rate of 12.7%. Drug release profile fit to Higuchi equation as M=91.836 +17.413t^1/2 with r being 0.9934. The total release of drug was 60% in 90 days. NGF kept more than 80% of its activity within 10 days, and then went through a relatively fast decrease. About 40% of the original bioactivity was kept on the 28th day and only 10% left on the 90th day. The bioactive NGF release profile was also fit to Higuchi equation: M=92.577+ 8.989t^1/2, r=0.9915. When 15% of additives was added to PLA, the strength and permeability of the PLA conduits was good. The compound conduit C (PLA conduit with 5 mg microspheres) was chosen as the appropriate compound conduit according to the regenerated results. After 15 days, the length of regenerated nerve was 9.2±1.66 mm in autogenetic nerve transplantation, 4.3±1.82 mm in the compound conduit with NGF microspheres, 1.5±1.53 mm in compound with physiologic saline and 2.3±1.44 mm in compound conduit with NGF solution, respectively. The length of regenerated nerve in the compound conduit was statistically different from the other groups (P<0.05). The structure of the regenerated nerve tissue was better in compound conduit group and transplantated autogenetic group.CONCLUSION: The characteristic of the microspheres could achieve design request when OVA was used as the protective agent and PLA:PLGA (1:1) was used as encapsulation material. The compound conduit with NGF microspheres could promote early nerve regeneration from the point of the length and quantity of the regenerated nerve fiber. The compound conduits with NGF microspheres had a better effect in promoting nerve regeneration than those with NGF solution or physiologic saline, but somewhat inferior to transplanted autogenetic nerve.