Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neurons Induced By Self-assembly Of A Hydrogel From A Branched Peptide Amphiphile

Purpose:Explore the co-culture of bone marrow mesenchymal stem cells(BMSCs)and dendritic amphiphilic polypeptide(B-PA)self-assembled hydrogels,observe cell biocompatibility,cell growth status,cell adhesion performance and induce bone marrow The differentiation of mesenchymal stem cells into neuronal cells.It provides theoretical feasibility for hydrogel stent implantation to treat patients with spinal cord injury.Method:1.Use whole bone marrow adherence method to isolate and culture bone marrow mesenchymal stem cells(BMSCs),observe cell differentiation and morphological changes with an inverted phase contrast microscope,identify bone marrow mesenchymal stem cells using flow cytometry,and select 3-4-week-old SD(Sprague Dawley).2.Use Solid-phase peptid synthesis(SPPS)to synthesize IKVAV(I-isoleucine,K-lysine,V-valine,A-alanine,and V-valine).Adenine)and RGD(R-arginine,G-glycine,D-aspartic acid)linked dendritic amphiphilic peptides,and use high-performance liquid chromatography(HPLC)and mass spectrometry(MS)to analyze the Characterization.Prepare a 1% mass fraction B-PA solution and add an equal volume of DMEM/F12 for a few seconds to self-assemble into a translucent hydrogel,and use transmission electron microscopy(TEM)to observe the internal structure of the self-assembled hydrogel.3.Use the dead and living cell detection reagents Calcein-AM and PI to double-label the co-cultured B-PA self-assembled hydrogel and bone marrow mesenchymal stem cells,and observe the bone marrow in the B-PA self-assembled hydrogel with a fluorescence microscope The survival of mesenchymal stem cells.The CCK-8 method can be used to detect the cell proliferation and cytotoxicity of B-PA hydrogels of different concentrations on bone marrow mesenchymal stem cells.The experiment was divided into three groups: containing PLL(polylysine),group IKVAV gel group,B-PA self-assembled hydrogel group,co-cultured with bone marrow mesenchymal stem cells with a density of 1000 cells/cm2.Hochest 33342 staining detects cell adhesion ability.4.After 7 days of culturing the B-PA self-assembled hydrogel and bone marrow mesenchymal stem cells,the cells were detected by immunofluorescence to differentiate into nerve cells expressing MAP-2,NF,and GFAP markers.Result:1.The subculture to the third generation of rat bone marrow mesenchymal stem cells was observed under an inverted microscope.The cell morphology was long spindle-shaped,spirally arranged,plastic-attached,and the subcultured cells grew fast.The flow cytometry result showed the ratio of CD29 positive cells.97.8%%,the ratio of CD44 positive cells was 99.8%;the ratio of CD34 positive cells was 1.61%,and the ratio of CD45 positive cells was 1.71%.2.B-PA design sequence: C16H31O-K-RGD K-IKVAV.High performance liquid chromatography(HPLC)purified and detected the purity of the peptide to be95.8507%.Mass spectrometer(MS)measured the molecular weight of the synthesized B-PA to be MW 2191.72,which was consistent with its theoretical value.TEM results show that the B-PA self-assembled hydrogel is composed of nanofibers,which cross each other in a network shape.The diameter of the nanofibers is 6～8nm and the length is between 10～500nm.3.Place the bone marrow mesenchymal stem cells into 1% mass fraction B-PA self-assembled hydrogel and culture for 1 day,3 days,and 7 days later,stain with Calcein-AM /PI(Live and Dead Cell Staining).The live and dead cells were counted under a microscope,and it was found that with the increase of time,the number of live cells increased significantly,and the dead cells decreased significantly.Use a microplate reader to detect the absorbance of CCK-8 at a wavelength of 450 nm.Groups A,B,C,and D all have an increasing trend in 1,3,and 7 days.The increase in the optical density(OD)value is related to the incubation time and B-PA concentration is positively correlated.There was no significant difference in OD values between groups A,B,C and D(P>0.05).However,group E has the lowest OD value.The PLL(polylysine),group IKVAV gel group,and B-PA self-assembled hydrogel group were co-cultured with bone marrow mesenchymal stem cells with a density of 1000 cells/cm2.After washing at 2h,4h,and 6h,the number of cell adhesion in all three groups(P<0.05)increased with time,and reached the peak in the B-PA group.Also,the number of adherent cells increased significantly between 2 to 4hours.4.After inoculating bone marrow mesenchymal stem cells on B-PA self-assembled hydrogel and incubating for 7 days,using immunofluorescence microscopy,the cells expressing MAP-2 and NF show red fluorescence,and the cells expressing labeled GFAP show green fluorescence.It shows that B-PA hydrogel can induce bone marrow mesenchymal stem cells to differentiate into neurons and glial cells.In conclusion:1.B-PA self-assembled hydrogel has good cell compatibility,promotes cell proliferation and adhesion,and is easily modified.It is worthy of recognition;2.B-PA hydrogel can induce bone marrow mesenchymal stem cells to differentiate into neurons and glial cells.