Induction Of Bone Marrow Stromal Cells Into Neuron-like Cells By The EGF-bFGF-chitosan Microsphere

Background and objectiveStudies confirmed that bone mesenchymal stem cells (BMSCs) may differentiate to neuron-like cells under inducement of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and had the effect of promoting spinal cord injury (SCI) recovery. But because EGF and bFGF may diffuse, transform or enzymolyze after entering human body, it can not retain its biological activity and can hardly achieve the effect of induce bone mesenchymal stem cells to differentiate to neuron-like cells. Applying chitosan sustained release microsphere loading cytokines can achieve the effect of resisting human body metabolism. The objective of this experiment is to prepare chitosan sustained release microsphere loading EGF and bFGF, and apply in BMSCs under culture in vitro, observing:1. The influence of EGF-bFGF-chitosan sustained release microsphere on activity and multiplication capacity of BMSCs.2. The condition that BMSCs is induced to differentiate to neuron-like cells and provide theory evidence for transplantation in vivo treating SCI by combining BMSCs, EGF-bFGF-chitosan sustained release microsphere.Methods1.In vitro culture and identification of bone marrow stromal cells.BMSCs from adult SD rats’femur and tibia were cultured by adherence screening method, and BMSCs stem cell markers CD44-CD45, bone marrow hematopoietic stem cell marker CD45 were detected by immunofluorescence assay.2.F-bFGF-chitosan microsphere preparation and detectionELISA method was used to detect EGF and bFGF, and the drug loading rate, the release rate, the rate of microsphere packaging were calculated. Flow cytometry was adopted to test cell toxicity of blank chitosan microspheres on BMSCs.3.Observation of EGF-bFGF-chitosan microspheres inducing BMSCs differentiation into neuron like cellsThe follow groups were prepared:A.Control group, BMSCs+DMEM;B.Blank chitosan microspheres group, BMSCs+DMEM+blank chitosan microsp-heres;CEGF/bFGF direct administration group, BMSCs+DMEM+bFGF (final concentra-tion 10ng/ml)+EGF (final concentration 20ng/ml); D.EGF-bFGF chitosan microspheres group, EGF-bFGF chitosan microspheres cell (make the concentration the same with that of group C);Changes of BMSCs cell morphology were observed under light microscope, MTT was used to detect the growth and proliferation condition of BMSCs, and the staining condition of neural precursor cell marker Nestin, neuron marker NSE and astrocyte marker GFAP were detected by immunofluorescence staining.Results1.The primary cultured bone marrow stromal cells were confluent at 10-14d, and the cells were in long spindle shape. After 4-5 passages, cells were with high purity, and mainly existed in elongated spindle BMSCs shape. The fifth generation of BMSCs in vitro were identified by cellular immunofluorescence method, and cells were observed positive expression of CD44, CD90 and CD45 presenting negative expression.2ulsification chemical-crossline method for preparation of sustained release microspheres. Through ELISA detection, we knew that:when the final concentration of chitosan was 2 mg/ml, the average drug loading was the highest; and packing rate increased gradually with the increase of TPP concentration, when TPP concentration was 0.45 mg/ml, the encapsulation rate reached the peak. When EGF and bFGF were in PH7.4 and PH5.8 solvent, there was a burst release process about 40-60% of EGF and bFGF in 24h, and then the slow release began.3.The flow cytometry combining Annexin V-FITC/PI double staining was adopted to detect apoptosis condition of cells. It was showed that cell apoptosis rate had no significant difference (P> 0.05) between the normal control group and the treatment group treated with chitosan sustained release microsphere for 24h.The growth and proliferation condition of BMSCs was tested by MTT method. The comparing results of BMSCs cell survival rate from four groups showed that: compared with the control group, the cell proliferation rate of blank chitosan sustained release microspheres group was not statistically significant. The cell viability of EGF/bFGF group and EGF-bFGF-chitosan sustained release microsphere group was evidently incrased that that of control group and blank EGF-bFGF-chitosan sustained release microsphere group (P). MTT assay results indicated that blank EGF-bFGF-chitosan sustained release microsphere, as the carrier, had no significant influence on the survival of BMSCs. And EGF/bFGF group and EGF-bFGF-chitosan sustained release microsphere group can promote growth and proliferation of BMSCs. Annexin V-FITC/PI double staining results indicated that blank chitosan sustained release microspheres had no biological toxicity. MTT assay results indicated that blank chitosan sustained release microspheres, as a carrier, had no significant influence on the survive of BMSCs. EGF/bFGF group and EGF-bFGF-chitosan sustained release microsphere group can promote the growth and proliferation of BMSCs.4.Four groups after 5 days’BMSCs inducing culture, blank chitosan sustained release microsphere group and the control group kept unchanged, still maintaining the typical form of BMSCs; some cells of EGF/bFGF direct administration group and EGF-bFGF-chitosan sustained release microsphere group presented partially neuron like change. The HCS immunofluorescence:EGF/bFGF and EGF-bFGF-chitosan sustained release microsphere group:Nestin, NSE, GFAP fluorescent staining all have positive expression. Confocal immunofluorescence identification:(36.51±2.53)%of the cells expressed neural precursor cell marker Nestin, (17.66±3.44)% neurons marker NSE, and (27.39±3.03)% of the cells expressed the astrocyte marker GFAP; EGF/bFGF sustained release microspheres group:(30.87±3.53)% cells expressed neural precursor cell marker Nestin, (10.57±2.24)% cells neuronal markers NSE, and (21.31±4.69)% of the cells expressed the astrocyte marker GFAP.Conclusion1.Adherent method for rat BMSCs culture is a simple method, by which a large number of high purity BMSCs can be quickly got.2.Preparation of EGF-bFGF-chitosan microsphere by emulsion crosslinking method has the advantages of simple process, high encapsulation efficiency, large drug loading, good release properties in neutral PH value,and less biological toxic effects on BMSCs; besides, ti can also promote growth and proliferation of BMSCs.3.EGF-bFGF-chitosan microsphere can induce marrow stroma cell to differentiate into neuron-like cells.4.The efficiency that EGF-bFGF-chitosan microsphere induce BMSCs to differentiate into neuron-like cells is lower than direct EGF/bFGF, and may relate to the low primary concentration of EGF-bFGF-chitosan microsphere releasing cell factor. The best concentration that EGF-bFGF-chitosan microsphere induce BMSCs differentiation still needs further detection.