Experimental Study On Bridging Repair Of Recurrent Laryngeal Nerve In Rats By PLGA Based NSC-SC Tissue-Engineered Nerve Concuit In Vivo

Part 1 Co-cultured Schwann Cells and Neural Stem Cells In Vitro Experimental StudyObjective: To investigate whether co-culture Schwann cells(SCs) with Neural Stem cells(NSCs) improve the survival rate, direction of differentiation, and the secretion of BDNF, GDNF in NSCs.Material and Methods: Passage2-4 SCs and passage2 NSCs were grouped as follow: SCs alone, NSCs alone, and co-culture of SCs and NSCs. Observations of cellular morphological changes and quantification were both performed under an inverted phase contrast microscope. Immunofluorescence staining technique was used to identify cells: S100 for SCs, Nestin for NSCs, Map2 and Neu N for neurons, GFAP for astrocytes. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Enzyme linked immunosorbent assay(ELISA) was used to quantify the secretion of BDNF, GDNF factors in 1, 3, 5 and 7 days.Results: Cells in the Co-cultured group had a better state, with more NSCs differentiated into neurons. Cell survival rate and secretion of BDNF、GDNF in co-cultured group are higher than the other two groups(p<0.05).Conclusion:Co-cultured SCs and NSCs promote the differentiation of NSCs into neurons and can secrete more neurotropic factors(BDNF, GDNF), thus improving the microenvironment for nerve regeneration.Part 2 Construction of tissue-engineered artificial nerve in vitroObjective: To explore the feasibility of in vitro experiment of tissue-engineered artificial nerve by comparing the co-culture of Schwann cells and Neural Stem cell with individual cell(Schwann cell or Neural cell) seeded with three different materials.Material and Methods: Schwann cells and neural stem cells were co-cultured together with PLGA, chitosan-PLGA(c PLGA) and laminin/chitosan-PLGA(lc PLGA) in identical media. And a newly designed PLGA nerve conduit, coated with chitosan and(or) laminin with paralleled nanofiber filaments axially placed within the lumen was constructed. The biocompatibility and cytoxicity of co-culture of Schwann cells and neural stem cells, and individual cell was detected by MTT assays.Results: Result:After 7 days in culture, scanning electron microscope(SEM) observation revealed that the PLGA nerve conduits coated with chitosan plus laminin peptides had more parallel neurite growth than was observed for the other two materials. In the group with co-cultured Schwann cells and neural stem cells, longer neurites and more bridges between the cellular processes were observed by phase contrast microscopy.Conclusion: lc PLGA has good biocompatibility and no cytotoxicity, and that the axial nanofibers in the lumen guide the growth of neurites in a particular direction. Schwann cells and neural stem cell co-culture provided a mutually beneficial effect. These findings provide a biological basis for the future application or transplantation of this artificial construct in peripheral nerve injury repair.Part 3 Experimental Study on bridging repair of recurrent laryngeal nerve in rats by PLGA based NSC-SC tissue-engineered nerve conduit in vivoObjective: To study in vivo experiment of tissue-engineered PLGA-NSC-SC three-dimensional nerve conduit in repairing recurrent laryngeal nerve defects of rats, and test the functional effect by morphological, molecular biological and electrophysiological method.Material and Methods: Seventy two adult SD rats(weighing 150g)were randomly divided into 6 groups(n=12),and their recurrent laryngeal nerves(1ength:10 mm)were resected.In all groups except E and F group nerve defects were repaired using the 3D PLGA nerve conduit with different components, in which the nerve conduits in group A were filled with 3.75μl Schwann cells and 3.75μl Neural Stem cells, the one in group B filled with 7.7μl Neural stem cells, the one in group C filled with 7.7μl Schwann cells, and the one in group D filled with 30 μl matrigel. The rats in group E were subjected to nerve autograft(control group), while the rats in group F subjected to pseudo surgery.To evaluate the repairing outcome,middle segment of regenerated nerve was cut, and sections were used for immunofluorescence staining. And electronic microscopic evaluation was also done to study the morphological changes of regenerated nerve cells. EMG tests were also performed with the calculation of their latent period and amplitude of action potential in regenerated nerves of rats 8 and 12 weeks after operation.Results: Immunofluorescent staining test shows that regenerated nerve segments in CO group had a large amount of NF immunoreactive fibers and S-100 immunoreactive fibers surrounding the nerve fibers, which indicates that myelinated nerve fiber regeneration was successful. Under electronic microscope,it was found there was significant difference in types of cells,thickness of myelin sheath among different groups, in which there were a larger number of regenerated nerve fibers with a thick myelin, and less connective tissue between the beams, while the regenerated myelin had matured well with consisting thickness, and regenerated axons also developed well and were arranged in order in CO group, both 8 weeks and 12 weeks after surgery. Twelve weeks after operation,compared to groups B,C,D and E,the latent period was significantly decreased in group A(P<0.01), while there were no significant difference among different groups regarding results of amplitude. And, for the part of 8 weeks after operation, the latent period was also decreased in group A, compared with group B and C, but without statistical difference while.there was still no significant difference in amplitude of action potential among groups A,B,C,D and E(P>0.05).Conclusion: Tissue-engineered PLGA-NSC-SC three-dimensional nerve conduit may promote the regeneration of recurrent laryngeal nerve.