Basic Application Study Of The Tissue-engineered Nerve Conduits For Repairing Peripheral Nerve

Peripheral nerve defects are relatively common clinical disease. There are about one hundred million people getting peripheral nerve injury all the world each year. It would seriously affect the patient’s physical and mental health and decline their life quality. The nerve regeneration and maximize functional recovery after defects has been a major problem for the basis of neural scientific and clinical research at home and abroad in the past century. As nerve repair "gold standard", autologous nerve grafting has limitations, such as the limited sources. Which severely restrict its widespread use in the clinic. The developing of tissue engineering opened a new direction for finding the ideal alternative to nerve. In recent years, with the development and progress of tissue engineering, it opens up a new direction to find the ideal alternative for nerve regeneration.In this study, outer catheters are prepared with collagen/PCL by electrospinning. Multichannel conduits of the collagen composited with salidroside microspheres were made in a special mould. They were merged and crosslinked with 1% genipin to build a new type of tube in tube nerve conduit. The conduit scaffolds were analyzed by mechanical properties and the physical and chemical properties.Catheter core material composited with salidroside microspheres was vaccinated rat bone marrow mesenchymal stem cells(BMSCs) and cultured for different time. Cell adhesion was observed by scanning electron microscopy. Cell proliferation was measured by CCK-8 method. The differentiation of BMSCs into neural cells was measured by immunofluorescence assay.Wistar rat sciatic nerve 15 mm defect was made in animal experiments. Rats were divided into five groups randomly. They were hollow conduit group, blank material conduit group, composited with salidroside microspheres conduit group, autogenous nerve group and nerve defect group. There were 12 rat every group. After operation one month, two months, three months, footprint of each group was collected and measured sciatic functional index, the dates were statisticed in Oneway analysis of variance by SPSS17.0 software. Five months after operation the rats were reexposed the sciatic nerve, tested electrophysiological;fixed specimens and cut sections soon after. Nerve injury repairing and regeneration was evaluated by histological staining(HE), S100, Neurofilament(NF), Glial Fibrillary Acidic Protein(GFAP) immunohistochemistry staining.The results showed that the highest sustained rate of salidroside microspheres was about 23% at 8h, and decreased slightly then and stabilized at(20 ± 1)% between 8 ~ 72 h. The salidroside microspheres have a good release effect. The contact angle of nerve conduit shell around crosslinked was 120°and 0°.The nerve conduit shell has highly hydrophilic after crosslinked. The permeability of the nerve conduit shell around crosslinked were(60.2 ± 3.4)% and(71.1 ± 5.1)%. There is no significant difference around crosslinked(P >0.05). Before crosslinked, maximum load of the nerve conduit shell was 21.5~ 25.8 N, breaking load was 12.3 ~ 15.7 N, after crosslinked maximum load was 40.7 ~ 42.9 N, breaking load was 28.2 ~ 33.1N. There is significant difference after crosslinked(P <0.05). Neural tube shell have a certain mechanical strength. The water absorption of collagen core around crosslinked was(645±54)% and(764±88)%. There is significant difference between them(P <0.05).Rat BMSCs adhered, growed, proliferated well on the collagen core composited with salidroside microspheres. Phalloidin immunofluorescence staining showed visible nerve-like cell morphology. S100 immunofluorescence staining showed positive expression. The core material composited with salidroside microspheres can induced BMSCs into neural cells by sustained release salidroside.After the operation one month, two months, three months,the footprint and SFI of rats showed that composited with salidroside microspheres group was similar to autogenous nerve group and was significantly higher than other groups. Electrophysiological test results showed that there is no significant difference(P > 0.05) between the group composited with salidroside microspheres and autogenous nerve group of the incubation period. And there is significant difference(P <0.05) with other groups. HE staining showed that composited with salidroside microsphere conduit group had significant regenerating axons and it was not obvious in hollow conduit group. Immunohistochemical staining results showed that S100, NF expression levels were enhanced in hollow conduit group, blank material conduit group, composited with salidroside microspheres conduit group and autogenous nerve group; on the contrary, the expression of GFAP protein in turn weakened. The repair effect of composited with salidroside microspheres group was better than hollow conduit group and blank material group and approached autogenous nerve group.Multichannel conduits of the collagen composited with salidroside microspheres have good water absorption,permeability,histocompatibility and certain mechanical properties. The nerve conduits composited with salidroside microspheres can promote the nerve regeneration and functional reconstruction. It has a similar effect with autologous nerve graft. It has good prospects for clinical application.