Experiments Study Of Chemical Extracted Acellular Nerve Allograft Wrapped By Human Placenta Amnion Supplementing With BDNF And CNTF-engineered Bone Marrow Mesenchymal Stem Cells Embedded In Fibrin Glue On Repaired Of Rat Peripheral Nerve Injury

It is an important task to promote peripheral nerve regeneration after injury in neuroscience. Bone marrow mesenchymal stem cells(BMSCs) is one kind of adult stem cells that have self-renewal and multilineage differentiation potential.It has recently been reported that BMSCs can differentiate into neural cells,opening new frontiers in therapy for nerve injury.In this study, we repaired of rat peripheral nerve injury by chemical extracted acellular nerve allograft wrapped by human placenta amnion supplementing with BDNF and CNTF-engineered bone marrow mesenchymal stem cells embedded in fibrin glue. We wanted to offer the experimental data for clarifying the mechanism and clinical application.Our experimental study were divied into four parts:Part Ⅰ:Separate culture and identify of MSCs(mesenchymal stem cells) in ratPart Ⅱ:Expression vector constrction of BDNF and CNTF and expressed in bone marrow mesenchymal stem cells Objeetive Study Separate culture and identify BMSCs. Methods derived from rat bone marrow were isolated and culture expanded efficiently in vitro and its biological features are detected. We selected suitable culture medium and FBS concentration,separated and purified the BMSCs with adherent method. Conclusions The morphology of BMSCs was observed with invert-microscope constantly. BMSCs’growth was depicted by flowing cytometry. Results showed BMSCs could be isolated and proliferated by adherent method in vitro. The appearance of BMSCs were fibroblast-like cells. Part Ⅲ:Preparation of chemical extracted acellular nerve allograft and human placenta amnionPart Ⅳ:Experiments study of chemical extracted acellular nerve allograft wrapped by human placenta amnion supplementing with BDNF and CNTF-engineered bone marrow mesenchymal stem cells embedded in fibrin glue on repaired of rat peripheral nerve injury Objective BDNF and CNTF-engineered MSCs has the potential to treat an array of degenerative neurologic disorders including peripheral nerve impairment. But the following questions must be identified in its practice. First is the functional and structural influence to recipients inner peripheral nerve by cells transplantation; Second is the distribution and survival of grafted MSCs. Third is that how long can the BDNF and CNTF-engineered MSCs maintain the expression of the target gene in peripheral nerve.Methods Successful culture BMSCs, surface markers of it identify by flow cytometer show CD44:93.25%、CD34:4.63%、CD45:8.01%, Expressed BDNF and CNTF was detected by immunohistochemistry display that positive expression rate reach to90%, Amplification product through electrophoresis of RT-PCR show band is positive expression of target gene.Results Building a successful BDNF and CNTF-engineered bone marrow mesenchymal stem cells。Conclusions BDNF and CNTF transfection can elevated survivals of grafted MSCs. Thus, BDNF and CNTF-engineered BMSCs can be used as vehicles to deliver the BDNF and CNTF gene. Objeetive:Preparation of chemical extracted accllular nerve allograft and human placenta amnion. Methods:Rat sciatic nerveswere chemically extracted by Sondellmethod, with detergents of Triton X-100and sodium deoxycholate, and by improvedmethod, with detergents of Triton X-200, sulfobetaine-10(SB-10) and sulfobetaine-16(SB-16). The acellular nerve allografts prepared were evaluated by HE staining, myelin staining.Results:The cells and myelinwere removed thoroughly and the nerve structures were preserved well in acellular nerve allografts prepared by improved method.Conclusion:This new chemical extraction procedure with detergents ofTriton X-200, SB-10and SB-16is an ideal paradigm to develop high-quality acellular nerve allograft. And this acellular nerve allograft is promising to replace the autologous nerve graft. Objective To investigate the effect of chemical extracted acellular nerve allograft (CEANA)wrapped with human amnion matrix membrane supplementing with BDNF and CNTF-engineered bone marrow mesenchymal stem cells embedded in fibrin glue on the peripheral nerve regeneration.Methods Thirty-five adult male SD rats were selected.The sciatic nerves were harvested from the five rats to prepare CEANA. The other30rats were used to establish the left sciatic nerve defect models of10mm and divided into6groups:autogenous nerve graft with fibrin glue(group A,n=5),CEANA graft with BDNF+CNTF/BMSCs(5×106)embedded in fibrin glue(group B,n=5),CEANA wrapped with human amnion matrix membrane supplementing with BDNF and CNTF-engineered graft with fibrin glue(group C,n=5).CEANA wrapped with human amnion matrix membrane supplementing with BDNF engineered graft with fibrin glue(group D,n=5).CEANA wrapped with human amnion matrix membrane supplementing with CNTF-engineered graft with fibrin glue(group E,n=5).CEANA wrapped with human amnion matrix membrane with BMSCs(5×106) supplementing with fibrin glue(group F,n=5).CEANA wrapped with human amnion matrix membrane supplementing with fibrin glue(group G,n=5).The right sciatic nerves were exposed as the controls.At2,4,6,and8weeks.the rat’s static sciatic index(SSI)was measured.The histomorphomctric assessment of triceps surae muscles and nerve grafts were evaluated by Masson staining,toluidine blue staining,and transmission electron microscope(TEM)observation at8weeks after operation.Results Incisions healed well in each group.At2,4,6,and8weeks,the SSI increased gradually in3groups.SSI in group C was higher than that in groups A and G at4,6,and8weeks after operation(P<0.05).SSI in group C was slightly higher than that in group D、E、F,but had no significant difference(P>0.05).At8weeks,the wet weight recovery rate of triceps surae muscles and fibers number of myelinated nerve were better in group D、E、F than in group A、Qbut worse than in group C,showing significant differences(P<0.05).The triceps surae muscle fibers area and myelin sheath thickness had significant differences between group C and group A、G (P<0.01),but there was no significant difference between group C and group D、E、F(P>0.05).Conclusion CEANA wrapped with human amnion matrix membrane supplementing with BDNF and CNTF-engineered BMSCs embedded in fibrin glue can improve functional recovery of peripheral nerve injury.