Repair Of Long Peripheral Nerve Defects By Neuronal Tissue Engineering Nerve Transplantation

Background and Significance:Peripheral nerve injury is common clinical damage, and the repair of peripheral nerve defects is a worldwide problem. Difficulty is Contradictions between the slow growth rate of nerve and the fast atrophy of effectors.The improvement of speed and quality of nerve regeneration is an urgent need to solve the repair of nerve injury. At present the treatment of peripheral nerve defect strategy is based on the theory of regeneration of peripheral nerve injury, by providing a channel, and creating a favorable micro environment for nerve fiber regeneration, and it is involved the application of Neural tube and cytokines, as well as the recent emergence of tissue engineering nerve. These treatments have been achieved gratifying results in animal experiments and clinical trials. But for a long nerve defect. But this recovery rate was not yet satisfied. Effectors of the degeneration is still very severe. In view of this, we have proposed a "neurons bridging to repair of peripheral nerve defects" the idea is that by neurons relaying, the time for the axons arriving to the effctors is reduced and the regenerations of effecors is slow down.To test this idea, this study started from the clinical utility, intended to construct neuronal tissue engineering nerve with bone marrow-derived neural tissue committed stem cells (NTCSC)-derived neuronal cells and allogenic acellular nerve, and repair the dog sciatic nerve injury. mechanisms and effectsof repairing peripheral nerve with neuronal tissue engineered nerve, aiming to provide a new approachof repairing peripheral nerve defects.Section 1 culture, induced differentiation and identification of bone marrow-derived NTCSC.Objective:To study separation, culture, identification of bone marrow-derived neural tissue committed stem cells (NTCSC), as well as induction and differentiation to neurons, the separation, culture, identification, and the induction and differentiation of neurons, and to discuss the possibility of neuronal cells as a source of seed cellsMethods:The cells are Harvested from dogs bone marrow, and first cultured in a serum culture medium for 7days. Later the cells were moved to a serum-free medium cantaining with BFGF and EGF. After the cells grow to form cells sphere. Disperse the cells sphere and continue to cuture the cells sphere. And this cells sphere is bone marrow-derived NTCSC. Let adherent neurosphere and use RA+SHH to induce the NTCSC to neuronal differentiation, as well as NRG1 to induce to Schwann cell differentiation. At different time observe cell morphology, growth, detect surface marker of cells.Results:The bone marrow-derived neural tissue committed stem cells (NTCSC) suspend, and grow spherically and proliferative capacity of cells is strong. NTCSCs express CXCR4-positive, Nestin-positive. After RA+SHH induction, the neurospheres adherent and expressβ-Tubulin-positive and NeuN-positive. With NRG1-induced, the neurospheres spread out, grow long And express S-100 positive.Conclusion:NTCSC has great expansion capability and have characteristics of neuralline, NTCSC can differentiate into neurons and Schwann cells, as a result NTCSCs is an ideal seed cells for tissue engineering nerve.Section 2 Construction of neuronal tissue engineering.Objective:Prepare a safe nerve scaffold of non-immune response, suitable bio-mechanical properties. Seed cells to nerve scaffold and Prepare tissue-engineered nerve. Transplant the nerve to the beagle's leg.Methods:6cm sciatic nerve were havested from died beagle dogs.The nerve were Rinsed with 0.3% SDS for 5 days and RNA enzymes, DNA enzyme for 2 days, and rinsed with distilled water Repeatedly for 5 days. The Acellular nerve graft were sterilizated by 60Co irradiation, and then conserved at 4℃. Induced Schwann cells and neuron-like cells were mixed into the cell suspension in accordance with the ratio of 1:1, and injected into the scaffold with micro-syringe. And now the neuronal tissue-engineered nerve is prepared.Results:acellular nerve scaffold is of complete shape, and did not contain cells. The Fibers of scaffold were continuous and of non-fracture. After the cultivation of cells, tissue engineering nerveare soft and plump. The nerves were well suture with the scatic nerves. The planted cells grew in the scaffold and migration, distribution was observed.Conclusion:The biomechanical properties of Acellular nerve graft is good, containing less cellular components. The Acellular nerve graft is a safe carrier for nerve tissue engineering.Section 3 the in vivo research of neuronal tissue-engineered nerve.Objective:To study the effect of repairing the scatic nerve 6cm defects with neuronal tissue engineering nerve and disscuss the feasibility of repairing the long segment of peripheral nerve defects with Bridging the neurons.Methods:Prepare 6 canine models of 6cm defects of two Hind legs scatic nerve. Neuronal tissue engineering nerve,schwann cell tissue engineering nerve and autologous nerve graft were transplanted into the 4 scatic nerve defects. Animal behavior were observed after the operations. After 13 weeks, EMG was examined. 4 sides neuronal tissue engineered nerve graft group underwent retrograde horseradish peroxidase. after killing all the animals, immunohistochemical stain, toluidine blue staining, transmission electron microscopy detection and protein content detection were taken.Results:The left lower limb of all three groups dogs can not move after operations. After 2 weeks, muscle atrophy were found in the limbs of three groups begin muscle atrophy. After 8 weeks the experimental group dogs began to react to acupuncture, and lower limbs can touch the ground walking. Sensorimotor all recovered. Autograft control group recovered after 10 weeks and Schwann cell transplantation group were recovered after 13 weeks.After HRP were injected Gastrocnemius muscle of neuronal tissue engineering Nerve Graft dog, blue clumps is visible in nerve graft. Immunohistochemistry showed that neuronal tissue engineered nerve graft in NeuN positive.Toluidine blue staining showed that the number of myelin sheath in neuronal tissue engineered nerve graft is more than Schwann cells in tissue engineering nerve group.Transmission electron microscopy revealed synapse formation in neuronal tissue engineered nerve graft. Coomassie blue detection of protein content showed that protein content of gastrocnemius muscle in the experimental group> nerve autograft group> Schwann cell transplantation group.Conclusion:Transplanted stem cell-derived neuron-like cells can survive in the peripheral nerve microenvironment and maintain neuronal function. Repairing peripheral nerve defects by bridging the neurons is better than the traditional neural tissue engineering and autologous nerve graft. And this method is feasible.