| BackgroundSpinal cord injury(SCI) is a frequently encountered trauma in clinic of Orthopedic department. With the developing of modern architecture and transporting trade, the incidence of SCI presents a increasing trend every year. It is reported that the incidence of SCI is 60/1,000,000 every year in china. SCI often results in long-lasting deficits, involving partial or complete paralysis and loss of sensation below the level of the injury site, for the reason that SCI leads to huge damage for society and economy, and SCI becomes one of the challenges of medicine in 21th century. Presently, prevention the secondly injury of spinal cord and protection of remaining Neurons are still the treatment strategies for SCI clinically. However, the prognosis is ugly because of limited regenerative ability of spinal cord, the glial scar barrier and lack of neurotrophic factors blocking the axons regeneration.Treatment strategies for spinal cord injury are two aspects at present, involving promoting axonal regeneration and overcoming regeneration barrier. In 1981, Aguayo had proven that axons have the pontentail of regeneration in some appropriate conditions. It is an important landmark in Repairing spinal cord injury. With the emergence of tissue engineering, repair of spinal cord injury made new progress. The basic model of treatment of SCI was "seed cells + biological molecules + biological scaffolds ." Research found that genetically modified Schwann cells can secrete a large number of nerve growth factors to induce the regeneration of neurons, cylindrical biodegradable scaffolds seeded with Schwann cell transplantation for spinal cord injury repair has also been made a certain effect .Obiective1. Primary culture method for Schwann cells is established in vitro to obtain a large number of high-purity Schwann cells within a short period of time.2. Producing a new type of three-dimensional bioabsorbable scaffolds.3. SCs are transduced with lentiviral vectors encoding homo sapiens neurotrophin (LV-hNT3) to expresse transgenes lasting in high level.4. Three-dimensional scaffords seeded with SCs is transplanted to Spinal cord hemisection site , promoting axonal regeneration and restoring spinal cord function. We aim to explore the role of novel 3D scaffold seeded with LV-NT3-transfected Schwann cells for spinal cord injury repair.Materials and Methods1. We introduced a modified protocol combining the explanting and assimilation to culture primary SCs. the sciatic nerve of the rats were harvested and the epineurium was separated, then the nerve was cut in 1mm3 segments, dissociated with 0.03% collagenase and 0.25% trypsin and incubated at 37°C for 12 min. The segments were placed in culture dishes with DMED+10% fetal bovine serum. 24 hours later arabinosylsytoxin was added to eliminate the fibrablasts. Then, the growth medium was changed every 2-3 days.2. The novel three-dimensional scaffolds were made by means of melt spinning, extension and weaving., then biocompatibility and biodegradability were tested preliminarily in vivo and in vitro.3. From genebank we got the serial of homo sapiens neurotrophin 3 gene, and PCR was used to amplify the hNT3 cDNA, and LV-NT3 stocks were produced by cotransfection of the vector, packaging, and envelope plasmids into 293T cells. the number of transducing particles was defined by infecting 293T cells and counting the number of EGFP-expressing cells after 48h. Lentiviral vectors consisted of pGC-E1-EGFP, pHelper 1.0 (gag/pol ),Helper 2.0 (VSVG).4.We measured the transfection efficiency of the LV-NT3 transduction to SCs. scaffold seeded with LV-NT3-transfected SCs were transplanted to the spinal cord hemisection site , the female SD rats with 200g weight were chosed to study. SD rats were divided into three groups randomly: hNT3-SCs + scaffold, SCs + scaffold and scaffold. BBB locomotion scores are examined to learn functional recovery at 2,4, 8 and 12 weeks after injury. Tissues in SCI sites were observed with H&E, immunohistochemistry staining and electronmicroscopy to identify the survival of SCs, the regeneration of fibres and scaffolds degradation, hNT3 expression was identified by Western Blotting test.Results1. SCs was identified by S-100 staining. The purity of the cultured SCs was determined by comparing the number of Hoechst-labelled nuclei with the number of S-100 immunoreactive cells under a microscope. The purity reached 95.2% for the primary cells , it was Consistent with the literature.2. The novel 3D scaffold was produced by way of weaving, We prepared new type of scaffolds whose outer diameter was 3 mm, and the diameters of the micro-catheters and the fine wires were 100μm and 25μm, respectively. The inner diameters of the micro-catheters were uniform in transverse sections and showed a spiral increasing arrangement in axial sections. The porosity of scaffolds was 68%, and the 3D scaffold possessed features with good biocompatibi1ity , it's degradation time was 3 months.3. Titers of LV-NT3 were expressed as transducing units (TU) per milliliter and concentrated stocks ranged on the order of 5×107 TU/ml.4.The results showed that transfection efficiency was high reaching 85% when the MOI value was 10. hNT3 expression was identified by RT-PCR test, it was significantly higher compared with the control groups5. From 2 to 12 weeks,The BBB locomotion scores of hNT3-SCs + scaffold group was the best in all (P<0.05), especially group A > group B, group B > group C (P<0.05). H&E staining revea1d the number of cells in the scalford decreases at 8 weeks compared with 4 weeks , and less cells could be seen and the novel 3D scaffolds were completely absorbed at 12weeks. The results showed that 12 weeks after transplantation, the SCs were still alive. Under fluorescence microscope, the slices were observed that there was the expression of EGFP fluorescence after 4 weeks, less after 8 weeks and even after 12 weeks. Anterogradely labeled and retrogradely labeled axons were observed going through the region of spinal cord injury after 12 weeks. hNT3 expression was identified by Western Blotting test. New synapse formation were observed in the injury site after 8 weeks with electronmicroscopy, the region of SCI showed that more clusters of myelin, the new-grown myelinated and non-myelinated nerve fibers increased significantly after 12 weeks.Conclusions1.With the modified protocol combining the explanting and assimilation , the purity reached 95.2% for the primary cells , the cultured SCs could be used for tissue engineering technique for SCI repair.2.The novel 3D scaffold was suitable for SCI repair by tissue engilneering technique with good biocompatibi1ity and biodegradability。3. The hNT3 cDNA were amplified , and LV-hNT3 were constructed Successfully, Titers of LV-hNT3 were concentrated , stocks ranged on the order 5×107 TU/ml.4. The transfection efficiency of SCs was up to more than 85% in vitro, RT-PCR tests confirmed that hNT3 gene expression increased significantly. SCs maintained a good biological activity living for 12 weeks. hNT3 sustainable and efficient expression in SCs was identified, the axonal and synaptic regeneration were found in SCI sites. Spinal cord function got partially restored.
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