Primary Study On Allograft Of Schwann Cells For Spinal Cord Injury In Rats With Tissue Engineering Technique

With the development of modern industry, transporting trade and architecture, the incidence of spinal cord injury (SCI) presents a trend of increasing every year. It is reported that in USA there are 10 thousand patients suffering from SCI every year, and in Shanghai the incidence of SCI is 13.7/1,000,000 every year. There are some common characteristic points of the SCI around the word. The first is high rates of neurological disfunction ( the rate of complete paralysis is 67%). Second, the cost of healing SCI is very high. Third is the low mortality (<5%). Finally, the victim of SCI is always young people. 40% among them is younger than 40 years old. So, SCI always results in huge damage of society and great loss of economy. Presently, prevention and cure the secondly injury of spinal cord and protection of remaining nerve cells are still the mainstay key points of dealing with SCI clinically. Detailly, methyl- prednisolone (MP) has been shown to be neuroprotective if administered within 8 h after spinal cord injury. And surgical decompression of sipinal cord has been proven to be favorable to preserve the neurological function. Then, physical exercise is need to elevate life quality. However, the effects of these measurements are little, so the prognosis is ugly. This is because limited regenerative ability of spinal cord, and the chemistric and physical barrier block the regeneration neuron. So, promoting regeneration and overcome the barrier is the hotkey all around the world for the neurologic scientists.In recently 20 years it has been proven that the matured central nerve has the pontentail of regeneration, and the injuried spinal cord can regenerate in some congruent conditions. In some basic study of promoting axonal regeneration, the mammiferous spinal cord has regenerative ability with several measurement such as cell transplantation, gene therapy, and so on. So, Schwann cell transplantation for spinal cord injury has been widely explored.Schwann cells possess several intrinsic features that contribute to successful axonal regeneration in the injured peripheral nervous system. Schwann cells 1) secrete growth factors, including NGF, brain-derived neurotrophic factor (BDNF), glial cell line– derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF); 2) produce and secrete molecules of the extracellular matrix, including laminin, to which injured axons can attach and extend; 3) guide regenerating axons in combination with other structural elements of the injured peripheral nerve; and 4) remyelinate regenerating axons. These features could be useful in promoting axonal regeneration in the injured central nervous system. Indeed, several previous studies have demonstrated that Schwann cells grafted to the central nervous system (CNS) as either cell suspensions or attached to artificial surfaces can remyelinate CNS axons and support some degree of axonal growth. Often, however, the degree of axonal growth detected within Schwann grafts in the CNS is modest, suggesting that alternative means are required to augment the suitability of the Schwann cell as a conducive medium for CNS axonal regeneration.Previously, it has been reported that primary fibroblasts transduced to produce and secrete neurotrophic factors can support extremely robust growth of neurotrophin-sensitive axonal populations in the spinal cord and, in some cases, can support partial functional recovery. Schwann cells are potentially superior to fibroblasts as targets for gene modification and grafting to the CNS; however, because Schwann cells are native to the nervous system, can secrete extracellular matrix molecules that support axon growth, and can remyelinate extending axons. By augmenting Schwann cell features that naturally support axonal regeneration with high and sustained levels of neurotrophin expression, it may be possible to elicit more substantial growth of axons in the adult CNS. To test this hypothesis, primary Schwann cells were transduced to produce and secrete human nerve growth factor (hNGF). Primary rather than immortalized Schwann cells were studied because primary cells can be allografted, eliminating the risk of graft rejection. Thus, primary-transduced Schwann cells were characterized in vitro for hNGF production, then grafted in vivo to the lesioned midthoracic spinal cords of adult rats. It has been shown to promote axonal regeneration and myelination. However, axons do not regenerate beyond the transplant due to the inhibitory nature of the glial scar surrounding the injury. To overcome the glial scar inhibition, additional approaches such as increasing the intrinsic capacity of axons to regenerate and/or removal of the inhibitory molecules associated with reactive astrocytes and/or oligodendrocyte myelin should be incorporated. Clearly, Schwann cells have great potential for repair of the injured spinal cord, but they need to be combined with other interventions to maximize axonal regeneration and functional recovery.Tissue engineering, as an emerging and rapidly growing field, has received extensive attention. The ultimate goal of tissue engineering as a treatment concept is to replace or restore the anatomic structure and function of damaged, injured, or missing tissue or organs following any injury or pathological process by combining biomaterials, cells or tissue, biologically active molecules, and/or stimulating mechanical forces of the tissue microenvironment. Biomaterials are fashioned into three-dimensional scaffolds to provide mechanical support, and guide cell growth into new tissues or organs. The scaffolds have to be highly porous to allow seeding of cells at high densities, and upon implantation into the body, to facilitate the infiltration and formation of large numbers of blood vessels for nutrient supply of the transplanted cells and the removal of waste products. It is obvious that the emergence of tissue engineering has created an air of enthusiasm and optimism for the hundreds of thousands of patients worldwide who suffer from nervous system injury. Studies now indicate that some degree of CNS anatomical regeneration and functional recovery is possible following traumatic spinal cord injury.In present study, we culture primary Schwann cells from the sciatic nerve of the newborn SD rats. And SCs are transduced with lentiviral vectors encoding hNGF (LV-hNGF) to expresse transgenes in high level for long time. Then, transplant the reabsorble scafford seeded with transduced SCs to the acute spinal cord injury to promote axonal regeneration. Our goal in this study is to explore a promising strategy to induce directed nerve regrowth following spinal cord injury.In the first part of the study, we introduce a modified protocol combining the explanting and assimilation to culture primary SCs. Briefly, the sciatic nerve of the rats were harvested and the epineurium was separated and the nerve was dissected into discrete fascicles. Fascicles subsequently cut in 0.5mm3 segments, then dissociated with 0.03% collagenase and 0.25% trypsin and incubated at 37°C for 12 min. The segments were then washed with DMED+10% fetal bovine serum and placed in 30mm culture dishes incubated at 37°C in 5%CO2. 24 hours later arabinosylsytoxin was added to eliminate the fibrablasts. Then, the growth medium was changed every 2-3 days. The results shows that in the first 6 days thd SCs grow quickly and at 12th day they reached confuency. The SCs were 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 95.1% for the primary cells and 96.3% for the second generation. With this modified protocol we can get 3.0╳10~6 cells by ten rats. Also, the SCs began to die 28 days later. So, the SCs cultured with this protocol can be used for tissue engineering technique for spinal cord injury.In the second part of this study, a recombinant Lentivirus expressing the hNGF and GFP gene was prepared. From genebank we get the serial of the human NGF gene, and PCR was used to amplify the hNGF fragment, which contains the signal sequence required for the release of human NGF. hNGF fragment was cloned into pRRLsin-PPThCMV- MCS-wpre and LV-NGF stocks were produced by cotransfection of the vector, packaging, and envelope plasmids into 293T cells. After 2 days, medium with viral particles was harvested and, if needed, was concentrated by ultracentrifugation. For LV-GFP stocks, the number of transducing particles was defined by infecting 293T cells and counting the number of GFP-expressing cells after 48 h. Titers were expressed as transducing units (TU) per milliliter and concentrated stocks ranged on the order of 108 TU/ml.In third part of this study, we measure the infection effect of the LV-NGF transduction to SCs. When SCs reached confluence, replication-deficient LV-NGF-GFP vectors were added to the dish at a multiplicity of infection of 1:1, 1:5, 1:10, 1:20, 1:50 each other for 48 h. Expression of the reporter gene GFP was visible directly under a fluorescence microscope. The results showed that infection effect was highest reaching 90% when the MOI value was 1:10. Immunoreaction with anti-NGF antibody was carried out to reveal NGF-positive SCs 7 d after transduction. NGF expression was identified by the by antibody with RT-PCR and Western Blot test.In the fourth part of the study, SCs were prepared as described in the first three parts, and transplantation with scafford to the spinal cord was performed in vivo. The female SD rats with 150-200g weight were chosed to study. After opening the skin and muscle layers, a laminectomy was performed at the T8–9 vertebral level and the dura mater carefully opened. The exposed T9–10 spinal cord was transected and 3mm oftissue including visible spinal roots completely removed. After hemostasis was achieved, a 4mm long glu sponge scaffold with GFP-NGF-SCs solution was implanted in between the rostral and caudal spinal cord stumps. Every rat was implanted 2×10~6 cells averagely. The muscle layers were closed separately and the skin closed with metal wound clips. The rats received 10 ml Ringers'solution subcutaneously and were placed in warmed cages with food and water readily available. To prevent urinary tract infection, penicillin was administered daily during the first 7 days post-surgery. The bladders were expressed manually two times a day until voluntary bladder release returned. The rats were euthanized with a lethal dose of pentobarbital sodium and T8-9 spinal cord tissue was collected 1, 4 and 8 weeks respectively after transplantation. SCs survival, NGF expression and the scafford reasorbable were measured. The results showed that 8 weeks transplantation, the SCs were still alive and the NGF was expressed. The glu sponge began to degrade 4 weeks after transplantation. And 8 weeks later, most of the scafford degraded. From this part, we can conclude that it is feasible to cure SCI with tissue engineering tenique we described before.In the current study, the potential therapitic method using NGF gene modified SCs and glu sponge scafford was assessed through a broad array of tests. In vitro, we explored a modified protocol to culture primary SCs and found out their growth rule. We constructed the recombinant LV-NGF and test its infection efficiency to SCs. In vivo, the biocompatibility of SCs and glu sponge was tested and SCs survival and transduction gene expression was tested. However, in the present study the neurological functional improvements of the hind limbs after implantation and the axonal regeneration were not tested in this study. So, in the future study, we will emphasize on the study of axonal regeneration and functional improvements using this tissue engineering technique we introduced.