| The study aimed to prepare Alginate-polylysine-Alginate (APA) microcapsules containing nerve tissue of rats , and prepare acellular nerve with chemical extraction methods, to explore the effects of transplantation with microencapsulated peripheral nerve tissue and acellular nerve for different longitudinal sciatic nerve defects in dogs, therefor, to explore the difference between recellular acellular nerve and acellular nerve in regenation of nerve defect. It would lay a foundation for a new alternative treatment plan to substitude autograft for peripheral nerve defect.Part 1 Construction of acellular nerve of dogsObjective: To prepare acellular nerve of dogs and evaluate the biological features after imbeded into the muscle of rats'back.Methods: The sciatic nerves were collected from dogs weighed from 10kg to 15kg. The acellular nerve were prepared by chemical extraction. After sterilization by gamma irradiation, the morphology of the acellular nerve segments was revealed by light microscopy and scanning electron microscopy. The porus diameter and porosity were explored by image analysis. Five Sprague-Dawley rats weighed from 450g to 500g, 1cm acellular nerve were embeded in muscle of back of every rat to evaluate the biocompatibility. The rats were sacrificed at 1week after embedment, acellular nerve segments were removed and histological changes were determined.Results: We found that extraction with the detergents Triton X-100 and deoxycholate resulted in acellular nerve segments with preserved basal lamina tubes. The cellular components were almost removed and immunogenicity was decreased. Such grafts reflected no excessive signs of inflammation after embedment 1 week.Part 2 Prepare microencapsulated peripheral nerve tissue of rats and investigate its biological activityObjective: To prepare APA microencapsulated peripheral nerve tissue of rats and evaluate its biological activity in vivo and in vitro.Methods: The sciatic nerves were collected from rats weighed from 220g to 250g, and after trypsinization nerves became in cell tissure clump state. The APA microencapsules entrapping peripheral nerve with droplet generator were prepared and injected into the abdominal cavity of mice weighed from 20g to 25g. After embedment 6weeks microcapsules were reclaimed and recovery rate were caculated, microencapsulated peripheral nerve tissue wre cultured in vitro subsequentlly, its biological activity was evaluated with MTT and Elisa methods and the cell/tissure in the microcapsules were recultured.Results: We found that microencapsulated peripheral nerve could remain intact after transplantation at least 6 weeks, the recovery rate fluctuated from 80～90%, the peripheral nerve cells survived and the proliferation function of cells were maintained.Part 3 Study of the change of gastrocnemius bioactivity after transplantation with microencapsulated peripheral nerve tissue and acellular nerve for 3cm sciatic nerve defect in dogsObjective: To investigate the activity of muscle gastrocnemius after acellular nerve transplantation with microencapsulated peripheral nerve tissue repairing 3cm sciatic nerve gap in dogs.Methods: 24 dogs were selected and divided randomly into 3 groups: group A, group B and group C, each containing 8 dogs. The dogs of group A and B were transplanted by acellular nerves to bridge 30 mm sciatic nerve gaps, additionally in group B : microencapsulated peripheral nerve were transplanted too. Group C: autograft group. The motor function of operated-limbs were obserbed in regular after operation. At 6 months postoperative, The motor conducting velocities of the grafting segment were detected in all 24 dogs, both sides'muscle gastrocnemius were obtained and succinic dehydrogenase(SDH),synaptophysin(SYP) and cholinphospholipoidase histochemical stained , 1 cm distally sciatic nerve of distal end stoma were obtained and HE,Masson,Luxol fast blue,S-100 and NF200 stained.Results: We found that All the dogs of groupA,B and C were involved in the results analysis without loss. Image analysis indicated the optical density and the cross section area of muscle gastrocnemius,area and optical density of synaptophysin,area and optical density of cholinphospholipoidase in group B were not as statistic significant as in group A and similar to group C. The advancement of motor function recovery of operated-limb,detection of electrophysiology and morphology detections of sciatic nerve were all consistent with the detections of muscle gastrocnemius.Part 4 Study of the change of gastrocnemius bioactivity after transplantation with microencapsulated peripheral nerve tissue and acellular nerve for 6cm sciatic nerve defect in dogsObjective: To investigate the activity of muscle gastrocnemius after acellular nerve transplantation with microencapsulated peripheral nerve tissue repairing 6cm sciatic nerve gap in dogs.Methods: 24 dogs were selected and divided randomly into 3 groups: group D, group E and group F, each containing 8 dogs. The dogs of group D and E were transplanted by acellular nerves to bridge 6cm sciatic nerve gaps, additionally in group B : microencapsulated peripheral nerve tissure were transplanted too. Group F: autograft group. The motor function of operated-limbs were obserbed in regular after operation. At 6 months postoperative, The motor conducting velocities of the grafting segment were detected in all 24 dogs, both sides'muscle gastrocnemius were obtained and succinic dehydrogenase(SDH),synaptophysin(SYP) and cholinphospholipoidase histochemical stained , 1 cm distally sciatic nerve of distal end stoma were obtained and HE,Masson,Luxol fast blue,S-100 and NF200 stained.Results: We found that All the dogs of groupD,E and F were involved in the results analysis without loss. Image analysis indicated the optical density and the cross section area of muscle gastrocnemius,area and optical density of synaptophysin,area and optical density of cholinphospholipoidase in group E were not as statistic significant as in group D and similar to group F. The advancement of motor function recovery of operated-limb,detection of electrophysiology and morphology detections of sciatic nerve were all consistent with the detections of muscle gastrocnemius.Conclusions:1 Morphological and biocompatibility assessments indicate that acellular nerve scaffolds prepared by chemical extraction are suitable for facilitating nerve repair.2 Microencapsulated peripheral nerve can remaining bioactivity at least 6 weeks in vivo, the aim of recellularize acellular nerve through transplantation with microencapsulated peripheral nerve tissure was demonstrated practicable.3 After transplantation with microencapsulated peripheral nerve tissue and acellular nerve for 3cm sciatic nerve defects in dogs, the muscle gastrocnemius can be again innerved by nerve and in relative shortly time, the muscle atrophy is significant decreased, the regeneration of nerve were better than transplantation only with acellular nerve, the effect of the latter seemed to be accepted reluctantly. There were not obviosly variability difference between the regeneration of nerve of group transplantation with microencapsulated peripheral nerve tissue and acellular nerve and group autograft. As for whether exist variability between long term generation of nerve of the these groups or not and the necessity of recellular for acellular nerve for longer nerve gaps need to be investgated et sequentes.4 After transplantation with microencapsulated peripheral nerve tissue and acellular nerve for 6cm sciatic nerve gaps in dogs, the muscle gastrocnemius can be again innerved by nerve and in relative shorter time than ransplantation only with acellular nerve, the effect of the latter seemed to hard to be accepted. There were not obviosly variability difference between the regeneration of nerve of group transplantation with microencapsulated peripheral nerve tissue and acellular nerve and group autograft.5 The homeochronous comparison between transplantation groups 3cm and 6cm indicated it is more necessary in longer nerve gaps, and the recellularization of acellular nerve through transplantation with microencapsulated peripheral nerve tissure was demonstrated safe and effective. |
