The Study Of Construction Of Tissue-Engineered Artificial Nerve With Small Intestinal Submucosa Compound Of Schwann Cells To Repair Peripheral Nerve Defect

It is always a difficult problem to repair long peripheral nerve defect clinically. Currently autografting is the most common clinical treatment method, although it has some obvious disadvantages, for example, limited amount and length of nerve for grafting, disfunction of donor site, mismatching, misorientation regeneneration, etc. Recently, serching for an approriate artificial nerve alternative to autografting through tissue engineering technique has been hot spot.Schwann cells (SCs) play a very important role in genesis, development, morphology and function maintanece of peripheral nerve, which have been used widely as seed cells in peripheral nerve tissue engineering. Besides seed cells, a kind of appropriate scaffold is key for fabricating tissue-engineered artificial nerve. Currently, a variety of materials have been tested to bridge nerve gaps, which can be separated into two categories: synthetic and natural materials. Synthetic materials include polymers; Natural materials consist of the vein, degenerated skeletal muscle, collagen, or gelatin among the others. Unfornately, the materials above have some disadvantages. Up to date, none of them has reached to the equivalent level of the use of autografts and applied in repairing long peripheral nerve defect clinically. Small intestinal submucosa (SIS) is a kind of natural extracelluar matrix material. Its structure is close to the sophisticated structure of natural connective tissue and has more affinity for cells. SIS contains neither vessels nor cells as a kind of biomaterial, it could not induce obviously harmful immunological rejection when implanted in vivo as the xenograft. Now SIS has been widely applied as a biomaterial scaffold for experimental and clinical study of repairing the lower urinary tract, bladder, vessel, tendon, ligament, bone, meniscus, abdominal wall, dura, fascia, etc. Encouraging results have been reported, so SIS possesses the good application prospect. We had repaired rat 1cm short sciatic nerve defect by pure porcine SIS and gained satisfactory results.This study firstly isolated, purified, identified and passaged neonate rat SCs in vitro. SIS was prepared, and growth factor detection and biological safety evaluation were undertaken. Then SCs were co-cultured with SIS in vitro and the biocompatibility was studied. The tissue-engineered artifical nerve conduit was fabricated using SIS as scaffold and SCs as seed cells, and applied in repairing rat 14mm sciatic nerve defect. Pure SIS conduit and autografting were as control groups. We evaluated and analyzed the recovery outcomes and mechanism by means of multiple methods and discussed the possibility of repairing long peripheral nerve defect by it.Firstly, the method of isolation, culture and purification of neonate rat Schwann cells in vitro was investigated. Schwann cells were initially isolated from peripheral nerve of neonate rats in vitro using enzyme digestion method. Cytosine arabinoside (10-5mol/L) was added to inhibit fibroblast growth after 24h. Fibroblasts were further removed by means of different-speed adherence method and attained purification of SCs. Bovine pituitary extraction (40μg/ml) was added in culture medium to stimulate prompt division and proliferation of Schwann cells. Then, a large amount of Schwann cells with high purity concentration of 97% could be obtained as seed cells. This method has advantages of simpleness, convenience, little time consuming and high cell purity.Secondly, preparation, detection of growth factors and evaluation of biological safety of SIS was done. SIS was obtained by firstly removing the tunica mucosa, serosa and tunica muscularis through mechanical erasion. Cells escaping and sterilization was further done through chemical method. Finally SIS was preserved by freeze drying and sterilization. Light microscope and scanning electron microscope examination showed the residual cells on prepared SIS were almostly removed, and the ultrostructure and thermal stability of collagen fibers were nearly not destroyed. SIS possessed three-dimensional structure suitable for cell growth and could be used as scaffold in peripheral nerve tissue engineering. Vascular endothelial growth factor (VEGF) and transforming growth factor (TGF-β) could be detected in SIS by ELISA, and the content of them is almost same to which was reported in the literature. Pyrogenic test showed animal temperature elevation was in normal range after injected with SIS leaching liquor. So there is no residual pyrogenic material. Skin stimulation test and implantation in vivo found no abscess and fistule formation. Histological section showed there was moderate inflammatory reaction of forign object exciation at the early stage of implantation. At the intermediate stage, inflammatory cells decreased, mainly including lymphocytes, and there was little inflammatory reaction and antigen reaction. Meanwhile, SIS degraded sequently. At 12 weeks, SIS was almost completely degraded and absorbed. No degeneration and necrosis of muscular tissue was seen. IgG hypotype in serum detection showed IgG1 hypotype in mouse serum increased obviously at 1w after SIS implantation, reached the maximum at 4w, decreased gradually and get close to the normal level at 12w. There was no great variation of IgG2a and IgG2b hypotype through the period of SIS implantation. The results confirmed that the prepared SIS possessed good histocompatibility and had no toxicity generally and locally. It can induce no obviously harmful inflammatory and immunological rejection to the body after implantation in vivo, and be safely used in repairing tissue defect in vivo.Thirdly, the prepared SIS and Schwann cells were co-cultured in vitro and biocompatibility was studied. The SCs suspension was seeded on the surface of SIS, then we investigated SCs adhension, proliferation, differentiation and cell cytoactivity, and SCs secretion activity of neurotrophic factors was also evaluated at different times. MTT confirmed SCs had good proliferation activity on the surface of SIS and SIS had no cytotoxic effect on SCs. It showed that SCs adhered and proliferated well on the surface of SIS by contrast phase microscope, histological section and SEM, growing on the edge of the material or invaded into the foramina of SIS and reaching confluency on the surface of surface of SIS after 5~7d. SCs divided and proliferated in three-dimensional fashion, demonstrating long olivary, triangular or long fusiform shape with obvious prominence. Furthermore, The SCs connected end-to-end with each other or aligned in clusters and the protein granules secreted on cellular surface were also showed. TEM showed SCs grew on the surface of SIS in good condition. The cell body demonstrated long fusiform, having microvilli on their surface in abundance. SCs adhered tightly to the surface of SIS, growing in multilayer fashion locally. A considerable number of chondriosomes and ribosomes around the nuclear were seen in cytoplasm. At the interface of SCs and SIS, plenty of the minute foot plates were observed to attach SIS tightly. Furthermore, ELISA measurement revealed that, cultured in combination with SIS, SCs secreted NGF-βand BDNF prosperously without obvious difference with the contrast group, the secretory volume increasing with the prolonged time. The results of RT-PCR demonstrated the NGF-βmRNA and BDNF mRNA were expressed well by SCs co-cultured with SIS. Meanwhile, it confirmed that at 3～5d after SCs were co-cultured with SIS, the density of SCs on the surface of SIS and secretory volume of cell factors were both high and it was the best period for implantation in vivo. The results demonstrated SIS had good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit utilizing SIS compound of SCs.Finally, SCs as seed cells and SIS as scaffold material were co-cultured in vitro to construct tissue-engineered artificial nerve for reparing 14-mm defect of rat sciatic nerve. Pure SIS conduit and autografting were as control groups. The recovery results and mechanism were analyzed and evaluated by means of gross observation, electrophysiology, histology, immunohistology, image analysis, ultrastructure, retrograde tracing, blood supply reestablishment, etc. The results showed the ulcer of affected limb healed at 16w after operation in the SIS compound of SCs group, the diameter and appearance of graft is similar to the proximal and distal segment of sciatic nerve without neuroma formation. Plenty of regenerative vascular net was evident on the epineurium. Electrophysiological examination showed nerve conduction velocity (NCV) and amplitude of compound action potential (AMP) were not significantly different with the autografting group. Histological examination revealed that the regenerative nerve tissue grew through the gap successfully and a great quantity of myelined nerve fibers with thick myelin regenerated, arranging regularly in bundles. Image analysis showed percentage of regenerative nerve tissue, density of myelined nerve fibers and thickness of myelin had no significant difference with the autografting group. NF-160/200 immunohistological staining manifested a large amount of positive regenerative nerve fibres, arranging orderly with good continuity, which was similar to autografting. TEM showed that regenerative nerve fibres demonstrated mature morphology, myelinated and unmyelinated fibres of different diameter distributed uniformly with distinct layers of myelin sheath. The weight of triceps muscle of calf approached autografting. Retrograde tracing showed plenty of positive sensory neurons in L3～6 dorsal root ganglion and motor neurons in the anterior corn of spinal cord labelled by true blue, the resulted verified the afferent pathway of regenerative sensory and motor nerve fibres was unobstructed. Blood supply reestablishment analysis by vessel perfusion by ink and radioisotope scanning confirmed local blood supply reestablished in good condition.The results confirmed that the tissue-engineered artificial nerve constructed by SIS compound of SCs could repair long peripheral nerve defect in rats successfully, and the outcome was similar to autografting. It might facilitate repairing peripheral nerve defect the mechanism of the special three-dimensional surface structure of SIS, SIS containing plenty of cell factors, being favorable for SCs adhesion, growth and secretion of neurotrophic factors, promoting microcirculation reestablishment, etc. It is promising to become the alternative to autografting to be applied in repairing long peripheral nerve defect clinically after further study and exploration.