| 【Objective】This study aimed to explore the impact of vitro culture on the functional status of activated Schwann cells(ASCs),through comparing the difference of neurotrophic factors expression between ASCs and non-activated Schwann cells(SCs),and further investigate the impact of ASCs and SCs on the differentiation of neural stem cells(NSCs)into neurons.To compare the difference of biocompatibility between ASCs and SCs cultured in electrospinning poly-caprolactone(PCL)scaffold,and observe cell distribution,proliferation and differentiation,through cultured NSCs or NSCs combined with ASCs on electrospinning PCL fiber scaffolds,and further explore the biocompatibility of electrospinning PCL fiber scaffold for the co-culture system.【Methods】ASCs was isolated from sciatic nerve,which was ligated for 7 days,in Wistar rats and SCs was isolated from normal sciatic nerve.Cells were cultured and passaged to the third generation,and total protein were extracted for Western Blot,which was used to compare the differences of neurotrophic factor in protein level between ASCs and SCs.The NSCs were isolated from the hippocampus of E14.5 rat embryos,then the identity and the differentiation ability of NSCs was observed by immunofluorescence staining.The ASCs and SCs conditioned medium was half change,which was used to induce the differentiation of NSCs,then the neuronal differentiation rate and axon length were observed.ASCs and SCs was 3D cultured in the electrospinning PCL fiber scaffolds separately,and cell proliferation was tested by CCK-8 assay,cell distribution was observed through crystal violet staining to find the best cell cultured density,cell adhesion and morphology were observed by laser confocal microscopy and laser scanning electron microscope was used to study the distribution of cells.NSCs alone or with ASCs cultured in electrospinning polycaprolactone scaffolds,and the proliferation of cells was tested by CCK-8 assay,the morphology and distribution of the cells were observed by laser scanning and confocal microscopy,and the differentiation of neurons and expression of ASCs MBP were also observed.【Results】1.The expression of BDNF and NGF in ASCs were higher than SCs(P<0.05),and the expression of NT-3 and MBP were lower than SCs(P<0.05).After 7 days induction,in ASCM group,the ratio of neuron differentiation was 36.06±7.04%,the ratio was 17.22±3.78% in SCM group and the ratio was 5.78±3.03% in M group.The percentage of neurons in ASCM group was higher than that the other two groups(P<0.05).The axon length was 161.33±67.44 μm in group ASCM,and the axon length of SCM group was 110.33±60.34 μm,and the axon length was 97.55±54.09μm in group M.The axons of ASCM group were longer than those of the other two groups(P<0.05).2.The average diameter of fiber in electrospinning PCL fibrous scaffolds was about 7.93±1.41μm.Crystal violet staining was used to figure out the best cell density: the results showed that cells could connect with each other when the density of cells was at 2×10^4/cm2,and a certain number of ASCs distribute along with the longitudinal axis of fibers;after S-100 immunostaining,observed with laser confocal microscope,and the results showed that cells were spindle type,which was consistent with crystal violet staining results;and CCK-8 experiment showed the proliferation of ASCs in the PCL scaffold was faster than SCs,and the OD number at 7th days was higher than that of SCs(P<0.05).3.NSCs cultured in electrospinning PCL fiber scaffolds for 7 days,and the CCK-8 assay results showed that OD value increased gradually,and proliferation was slow in the first 3 days and cell proliferation reached the peak at the 7th day.The results showed that after 7 days of differentiation in PCL scaffold,NSCs could differentiate into astrocytes,neurons and oligodendrocytes through βIII-Tubulin,GFAP and O4 immunofluorescence staining,observed by laser confocal microscope.Then after NSCs and ASCs were cultured on electrospinning PCL scaffolds 7 days,the results showed that ASCs could express MBP,NSCs could differentiate into neurons and neurons distributed around with those ASCs expressed MBP.【Conclusions】1.ASCs can maintain a certain activation phenotype under in vitro culture conditions when passaged to the 3rd generation,and promote the differentiation of NSCs to neurons.2.Electrospinning PCL scaffolds could form 3D system,and have good biocompatibility with ASCs,and the body of cells distributed along the fibers;3.NSCs combined with ASCs cultured in electrospinning PCL scaffolds could form 3D cultured system. |
