An Experimental Study On Compatibility Of Bone Marrow Mesenchymal Stem Cells And Polylactic Glycolic Acid Scaffold, And The Effects Of Both Composite Which Restore To Peripheral Nerve Injury

ObjectiveTo investigate separately the compatibility between BMSCs,differentiated neural-like cells and PLGA scaffolds in vitro and in vivo,and explore the effects of both composite which restore to peripheral nerve injury,and provide a solid foundation of research to repair neurogenic bladder caused by SCI and diabetes.Methods1.BMSCs of rat were isolated and cultured by adherence screening method in vitro, and the morphological characteristics,growth characteristics,special staining of the primary and passage BMSCs were detected by Microscopy. BMSCs,surface antigens expression were detected by Flow Cytometry (FCM),to explore the feasibility of BMSCs as the seed cells of nerve tissue engineering.2.The induction medium were used to induce BMSCs to form Neural-like cells,and promote them to differentiate further into specific nerve cells.The induction and differentiation of BMSCs were detected by immunohistochemisty and Fluorescence Microscopy(FM).3.In vitro,MTT assay was applied to investigate the information of BMSCs'proliferation on the PLGA scaffolds,and PI staining was applied to observed BMSCs'cycle by Flow Cytometry (FCM).Via Scanning Electron Microscope to observed the information of adhesion,growth of BMSCs and neural-like cells on the PLGA scaffolds.4.In vivo,bold scaffold,BMSCs-PLGA complex,and neural-like cells-PLGA complex were separately transplanted into SD rat models,with the injury for right sciatic nerve.To obsvered SD rat models during 60 days after operation,and the right lower extremity muscle strength and motor function of rat were measured by ramp test, the recovery of right sciatic nerve were detected by histological and Electromyography(EMG).Results1.Cells were cultured by adherence screening method,in accordance with BMSCs in the structure and characteristics,and express only the type of interstitial cell surface markers,but not lymphocyte surface markers or macrophages by Flow Cytometry,which can be seen green fluorescent form of BMSCs with the corresponding antibody staining under Fluorescence Microscope.2.BMSCs were suspended grow like spherical after the induction,which were Nestin-positive by immunohistochemisty method.There are a large number of cells move out from the Neural Stem Cells after BMSCs were induced by adding serum.It respectively available to Neurons cells of MAP-2 positive and Astrocytes cells of GFAP-positive by immunohistochemisty method.3.In vitro,MTT assay showed that BMSCs which growth on the PLGA have a good activity,the OD values of whom and cell in the cultured plate are no significant at the same period(P>0.05). It prompts the BMSCs on the PLGA and in the culture plate was no significant difference.Cell cycle analysis also showed that most cells in a quiescent,only a small number of cells in the proliferative phase,and mostly diploid.All of these proved that the cultured cells have characteristics of stem cells.Scanning Electron Microscopy revealed that BMSCs and neural-like cells which were induced and differentiated,are closely combined with PLGA.It indicates that they have a good biocompatibility.4.In vivo,visual observation showed that Cell-PLGA complexes with no obvious nervous tissue boundaries,and most of the PLGA had degraded;biopsy revealed that it has a large number of nerve fiber cells,no obvious scar formation in the primary defects of the neural tissue.It proveds that PLGA with the host nervous tissue has good biocompatibility;ramp test showed that the angle of neural-like cells-PLGA combined group higher than the other groups;EMG showed that the nerve conduction and muscle recovery of neural-like cells-PLGA combined group faster than the other groups,too.Conclusion1.It is feasible to dissociate and culture BMSCs by adherence screeing method.2.BMSCs have more potential to differentiate into neural-like cells,and neural-like cells differentiate further into specific nerve cells in vitro.3.BMSCs and neural-like cells can be cultured in vitro and in vivo showed that they are feasible for ideal seed cells of tissue engineering.4.PLGA is a good biocompatibility nerve scaffolds materials of tissue engineering, which is conducive to the seed cells to conglutinate,growth and differentiate.It can repair rat sciatic nerve injury,promote the recovery of lower extrerat sciatic nerve injury,and promote the recovery of lower mitymotor function after combined with cells.5. It is feasible to restore the bladder functions after SCI for tissue engineering materials combined with cells.