Laminin-Chitosan-PLGA Conduit Co-Transplanted With Schwann And Neural Stem Cells To Repair The Injured Recurrent Laryngeal Nerve

Objective: This study aimed to explore an improved method for quickly acquire a large number of highly-purity of Schwann cells,and assessed the possibility and evaluating the effect of Schwann cells combined with neural stem cells and laminin-chitosan-PLGA nerve conduit on recurrent laryngeal nerve defect repairs.Methods: Digest the sciatic nerves that isolated from 3-5d SD-rats by improved compound enzyme method(collagenase and neutral enzyme)to obtained primary Schwann cells.Then the prepared Laminin-chitosan-PLGA nerve conduit,culturedSchwann and neural stem cells,and micro-surgical techniques were used to repair the5mm–long recurrent laryngeal nerve defect.Functional and histological assessments were respectively performed 8 and 12 weeks after surgery.And the effect was compared with nerve conduit with Schwann cells group,nerve conduit with neural stem cells group,nerve conduit group,autologous nerve transplantation group,and sham operation group.Results: 1.A large number of highly-purity Schwann cells can be quickly obtained by improved complex enzyme digestion method.The purity of cells was more than 98%when passage two times.The improved culture method was better than the traditional method.2.12 weeks after surgery,in the aspect of electrophysiology and neurotrophic factorsecretion,the result of CO group was superior to AUTOGRAFT group,and in the aspect of the number of regeneration myelin,AUTOGRAFT group has better effect;CO group and AUTOGARFT group has no significant difference in regeneration myelin sheath area.PartⅠ The study of an improved rat Schwann cells culture methodObjective:This study aimed to explore an improved method for quickly acquire a large number of highly-purity of Schwann cells,and then the cultured cells were identified by Immunofluorescence staining.Methods: Digest the sciatic nerves that isolated from 3-5d SD-rats by improved compound enzyme method(collagenase and neutral enzyme)to obtained primary Schwann cells.The improved method was compared with traditional Schwann cells culture method.And then the cultured cells were identified by Immunofluorescencestaining.Results:1.A large number of highly-purity Schwann cells can be quickly obtained by improved complex enzyme digestion method.The purity of cells was more than 98%when passage two times.The improved culture method was better than the traditional method.2.Immunofluotescence staining identification of cultured cells showed that the specific markers of S-100 were positive.Conclusion:A large number of highly-purity Schwann cells can be obtained by the compound enzyme digestion method,and this method was superior to traditional Schwann cells culture method.PartⅡ Laminin-chitosan-PLGA conduit co-transplanted with neural stem cells and Schwann cells to repair the injured recurrent laryngeal nerveObjective:This study aimed at assessing the possibility and evaluating the effect of laminin-chitosan-PLGA nerve conduit combined with the co-transplantation of Schwann and neural stem cells on recurrent laryngeal nerve defect repairs.Methods: In this study,120 female Sprague Dawley rats were used for establish animal models of laryngeal nerve injury,and the rats were randomly divided into 6 groups for further experiment : Laminin-chitosan-PLGA+SCs+NSCs group(CO),pure Schwann cells group(SC),pure neural stem cells group(NSC),nerve conduit group(NULL),Autologous nerve graft group(AUTOGRAFT),sham operation group(SHAM).The Laminin-chitosan-PLGA nerve conduit,cultured Schwann and neural stem cells,and micro-surgical techniques were used to repair the 5mm–long recurrent laryngeal nerve defect.Functional and histological assessments were respectively performed 8 and 12 weeks after surgery.Results: 12 weeks after operation,well-developed myelin sheath could be observed in CO group under transmission electron microscope,the myelin sheath thickness and diameter is greater than those in AUTOGRAFT group,but myelin sheath area has no significant difference with those in AUTOGRAFT group.The number of myelin sheath could be counted under toluidine staining,and the result shoed that the myelin sheath number of CO group was more than AUTOGRAFT group.The result of EMG shows that the latency and amplitude recovery of CO group is better than AUTOGRAFT group.The Real-time q PCR result showed that the expression of BDNF and GDNF secrete by laryngeal muscle and regenerated nerve in CO group were higher than other groups.12 weeks after surgery,the arytenoid cartilage abduction recovered well,and the glottis could almost be closed completely,the situation was similar to AUTOFRAFT group,and better than other groups except SHAM operation group.Conclusion: Based on these results,it can be concluded that Laminin-chitosan-PLGA conduit co-transplanted with neural stem cells and Schwann cells could promote nerve regeneration.