| The peripheral nerve repair after injury have already been thought highlyamong people during the last decade. The Domestic and international scholarsmake use of the absorptive biomaterial or active material of nerve to promotenerve regeneration, but the methods were apart from the clinical application.Ideal nerve repair materials should be the bridge connecting to the injury nerve,in the meantime, provide active material of nerve. In this research we chosedtwo kinds of biomaterial-polylactic acid and polyglycolic acid, synthesizd to theartificial compound membrane (PLGA). Adjusting the qualities and proportionsof two kinds of materials of PLGA, make it degrade in 4-6 weeks. AddingVelvet antler polypeptides (VAP, the molecular weight 7200 Ds) extracted fromthe pilose antler and synthesizd to the velvet antler polypeptides-PLGA(VAP-PLGA) compound membranes (thickness 50 μm). After physical andchemical performance examination in vitro, it was used to wrap the sutures ofsevered sciatic nerves (D group) in rats, which were compared with shamoperation group (A group), model group (B group), blank membrane group (Cgroup) and mecobalamine-PLGA (E group). At 2, 4 and 6 weeks post-operation,general morphology, electrophysiology, histology, immunohistochemistry,·7·RT-PCR and nerve retrograde tracing observations and examinations wereinvestigated.Material and Methods(1) Physical and chemical performance examination to VAP-PLGAcompound membrane in vitro1. Analysis of release concentration of Velvet antler polypeptides inVAP-PLGA by ultraviolet spectra: Two kinds of different concentrations dosageof VAP-PLGA absorption peak A was examined by ultraviolet spectra, standardcurve was plotted according to absorption peak A corresponding differentconcentrations, we can get release concentration from absorption peak A ofsamples, accumulated release indicated the correlation of drug release andmembrance degradation.2. Examination of VAP in VAP-PLGA using SDS-PAGE: SDS-PAGE ofVAP and VAP-PLGA was performed to determine the change of moleculeweight of VAP.(2) Experiment on VAP-PLGA promoting nerve regeneration in vivoHealth male Wista rats , 250±50g in weight, were divided into 5 groups, 18each group. (sham operated): untreated after isolation of nerve;Group B (model):broken ends of nerve were anastomosed directly;Group C (negative control):broken ends of nerve were wrapped by PLGA. Group D (experimental): brokenends of nerve were wrapped by VAP-PLGA. Group E (positive control): brokenends of nerve were wrapped by mecobalamine-PLGA.1. After anaesthesia, ischiadic nerve was isolated about 20mm. Nerveswere not treated in group A;ischiadic nerve was cut 5mm from inferior borderof piriform muscles and then were anastomosed directly. In group C,D,E, 3mmischiadic nerve was cut, 5mm defect was made, cut was sewed up at 3,6,9,12 o'clock. PLGA,VAP/PLGA and mecobalamine-PLGA membrances were cutinto 10×10mm., broken ends of nerve were wrapped by them and fixed at theboth sides. intermediate space of membrances were sewed up thus made themcontaining ducts. Six rats was selected from each groups and examined afteroperations.2. Flare, uncles and its healing, muscles atrophy in left were abserved, andsciatic nerve function index (SFI) was calculated.3. Sciatic nerve operated was exposed and stimulated at distant and nearside of nerves, signal was received by gastrocnemius muscle, nerve conductvelocity and action potential amplitude was recorded.4. Adherence of stoma, degradation of nerve ducts, morphous ofregeneration and neuroma were observed. In groups B,C,D,E 10mm sciaticnerve was cut containing stoma, fixed by paraffin imbedding as usual, serialsections were made and HE staining.5. Using CIAS-1 automatic image analysis system, we examined neuraxisaccounts, regenerated axis section area, regenerated nerve section area, stomasection area, proximate nerve section area, remaining membrance thickness.And neuraxis regeneration ratio, maturation degree of regeneration exis, stomainflation.6. Immunohistochemistry was performed using ABC method. Theexpression of TGF-β, IGF was examined in sciatic nerve operation. The mean ofabsorbtion of nerve fibers postive staining was examined by image analysissystem.7. RT-PCR analysis of sciatic nerve. Total RNA was extracted from nervesamples, retrotranscription and PCR was performed, brightness of PCR productwas scanned and analyzed using gel image formation system.8. HRP retrogrssion tracing. Before 72h , In groups B,C,D,E ,30%HRP10μl was injected into sciatic nerve 4mm beneath stoma, spinal cord waistinflation and corresponding dorsal root ganglions (L3-L6) were selected afternerve cut, frozen section was made and developed by DAB reagents, medullatednerve fibers labelled by HRP were observed using light microscope.Result1. General morphology: The situations of wound healing, flare of ankle andtoe, muscular atrophy and SFI were excellent in A,D and E groups comparedwith B and C group. There were no rejections and inflammatory reactions ineach groups.Electrophysiology: The nerve conduction velocity (NCV) and waveamplitude (WA) in A, D and E group were better than those in B and C groups(P<0.01);C group were better than B group (P<0.01), and 6w post-operationwere better than 2 w ones (P<0.01).The consequence above all indicated that the sciatic nerve functionalrecovery in rats was very efficient in experimental groups.2. Light microscope: The medullated nerve fibers were rich in field of vision.There were more fibers and thicker in A, D and E groups than in B and C groups,and fibers in 6w post-operation were more than those in 2 w evidently, similar tothat of group A;Image analysis: There were normal neural axis in group A. The regenerationratios and degree of maturity of neural axis were higher in D and E group than inB and C group (P<0.01), and 6w there was more significant contras post-operationthan 2 w ones (P<0.01). The data above all were higher in B group than in Cgroup (P<0.01).The consequence above all verified that The regenerate ratios and degree ofmaturity of neural axis were high in D and E group, and high quality ofregenerated nerves was obtained.The ratio of section area of neural distal to proximal end was 1 in group A,which was approximated to 1 in C, D and E groups (P>0.05), and which wasexceed obviously 1 (P<0.01). The dates indicated that the PLGA membrane couldprevent nerve neoplasia.3. Immunohistochemistry and RT-PCR semiquantitative analysisLight microscope: The transforming growth factor-βantigen (TGFβ) andinsulin-like growth factor (IGF) stain in neural axis and myelin sheath wereweakly positivein A group, and medullated nerve fibers were rich in field ofvision. The transforming growth factor-βantigen (TGFβ) and insulin-like growthfactor (IGF) stain in regenerated neural axis and myelin sheath were deeper in Dand E group than those in B and C group, and 6w post-operation were deeperthan 2w ones. So the quantity and quality of regenerated nerve, in C group werebetter than those in B group.Image analysis and RT-PCR: in every phase point, the expression of TGF-βand IGF antigen in regenerated neural axis and myelin sheath were higher in Dand E groups than those in other groups, and especially in 6w post-operation(P<0.01). The expession of TGF-β and IGF in group B was the lowest of allgroups (P<0.01). but there were no differences in D group than E group(P>0.05).The nerve active factors can promote nerve regeneration by advancing theproduction of cylindraxile and myelin sheath. The results above all showed thatthe nerve active factors were highly expressed in experimental groups , so wecould draw a conclusion that VAP-PLGA membrane promote nerveregeneration via providing enough the nerve active factors and the effectivenessis positive correlated with secretion time and contents of active factors.4. Horseradish peroxidase (HRP) nerve retrograde tracing: in every phasepoint, there were plenty of medullated nerve fibers of spinal cord labeled by HRPin rats of A group. The medullated nerve fibers of spinal cord labeled by HRPappeared in C, D and E groups during the time 4~6 w, especially in 6w, and theHRP-positive labeled nerve fibers waere more in D and E groups than those in Cgroup. And it was found that there were no HRP-positive labeled nerve fibers ingroup B.The results above all proved that the quality of regenerated nerve inexperiment groups was the highest, and the tracer agent had passed through thenerves stoma.Conclusion1. The application of VAP-PLGA membrane to repair impaired peripheralnerves can prevent neural adherence and provid nerve active factors as well,thereby nerve regeneration is achieved. The effectiveness of VAP-PLGA is thesame to that of positive control group.2. The method of delayed release administration of to broken ends of nervethrough PLGA membrane change the traditional single means. and bring a newidea to the region administration.3. Depending on the time of repair injury nerve and the amount of medicinedelayed release to adjust PLGA membrane, we made full use of the role ofpromoting nerve regeneration of VAP.4. Compared with the traditional nerve duct, the duct made of VAP-PLGAmembrane can adjust the caliber on the basis of nerve sizes, which bring into fullplay promoting nerve regeneration through refraining from nerve crush andsecretory juice flow over.5. The velvet antler polypeptides (VAP) and the material of PLGAmembranes have been clinically applied many years, which are avirulent and noside effects, no function changes as well. So the VAP-PLGA membranes can beapplied to clinic for repair the injury nerves. |
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